Novel bispecific antigen binding molecules capable of specific binding to cd40 and to fap

ABSTRACT

The invention relates to novel bispecific antigen binding molecules, comprising (a) at least one antigen binding domain capable of specific binding to CD40, and (b) at least one antigen binding domain capable of specific binding to a target cell antigen, in particular Fibroblast Activation Protein (FAP), and to methods of producing these molecules and to methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from European Patent Application No.17164725.8, filed Apr. 4, 2017 and European Patent Application No.18158751.0, filed Feb. 27, 2018, the contents of which are incorporatedherein by reference in their entireties.

SEQUENCE LISTING

The present application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 8, 2018, isnamed P34214-US_SeqListing.txt and is 635,489 bytes in size.

FIELD OF THE INVENTION

The invention relates to novel bispecific antigen binding molecules,comprising (a) at least one antigen binding domain capable of specificbinding to CD40, and (b) at least one antigen binding domain capable ofspecific binding to a target cell antigen. In particular, thesebispecific antigen binding molecules further comprise (c) a Fc regioncomposed of a first and a second subunit capable of stable association.The invention further relates to methods of producing these moleculesand to methods of using the same.

BACKGROUND

Multiple molecular signals are required during the generation of apotent adaptive immune response. Signal one involves the binding of aT-cell antigen receptor (TCR) to its cognate antigen presented on thesurface of antigen-presenting cells (APCs). Signal two consists of theengagement of costimulatory receptors with their respective ligandsbetween T cells and APCs. One of the best studied and most importantcostimulatory effectors is the tumor necrosis factor receptor (TNFR)family member CD40 and its ligand CD40L (Elgueta R. et al., Immunol Rev.2009; 229(1):152-72). Several members of the TNFR family including CD40function after initial T cell activation to sustain APC and T cellresponses and thus have pivotal roles in the organization and functionof the immune system (Watts T. H. (2005) Annu. Rev. Immunol. 23, 23-68).The combination of different costimulatory TNFR family members allows asequential and transient regulation of APC and T cell activation andsurvival resulting in increased immune responses while maintaining tightcontrol of APC and T cell function. Depending on the disease condition,stimulation via costimulatory TNF family members can exacerbate orameliorate diseases. Activation or blockade of TNFR family costimulatorsshows promise for several therapeutic applications in multiple fieldsincluding cancer, infectious disease, transplantation, and autoimmunity.

Among several costimulatory molecules, the TNFR family member CD40 playsa key role in triggering immune responses by inducing maturation,survival, antigen presentation, cytokine production, and expression ofcostimulatory molecules of APCs, which then drive antigen-specific Tcell responses and NK cell activation by proinflammatory cytokines. CD40regulates immune responses against infections, tumors and self-antigensand its expression has been demonstrated on the surface of APCs such asB cells, dendritic cells (DCs), monocytes, and macrophages as well asplatelets, and cells of non-hematopoietic origin such as myofibroblasts,fibroblasts, epithelial, and endothelial cells (Elgueta R. et al.,Immunol Rev. 2009; 229(1):152-72). The CD40 ligand CD40L is expressed onactivated CD4⁺ helper T cells, platelets, monocytic cells, naturalkiller cell, mast cells, and basophils (Carbone E. et. al., J Exp Med.1997; 185(12): 2053-2060, or Elgueta R. et al., Immunol Rev. 2009;229(1):152-72). Expression of CD40 and CD40L is strongly upregulated inresponse to various immune stimulatory signals and CD40-CD40Linteraction between APCs and CD4⁺ T cells contributes to increased APCactivation and antigen-specific CD8⁺ T cell responses (Bevan M J., NatRev Immunol. 2014; 4(8):595-602). Similar immune stimulatory resultswere observed by using CD40 agonistic antibodies (Vonderheide R H andGlennie M J., Clin Cancer Res. 2013; 19(5):1035-43).

Engagement of the type I transmembrane receptor CD40 by its naturalligand CD40L, a type II transmembrane protein or by agonistic antibodiespromotes CD40 clustering and induces the recruitment of adapter proteinsto the cytoplasmic receptor domain. The recruitment of these adapterproteins known as TNF receptor-associated factors (TRAFs) leads tosynergistic activation of mitogen-activated protein kinases (MAPKs),phosphoinositide 3-kinase (PI3K) as well as canonical and non-canonicalnuclear factor κB (NFKB) signaling pathways (Elgueta R. et al., ImmunolRev. 2009; 229(1):152-72). In turn, this results in APC maturation andactivation, which then maximizes antigen-specific T cell responses.Recent studies have shown two different modes of action of agonisticCD40 antibodies in harnessing anti-tumor immunity. Beside its indirectmode of action by mediated tumor cell killing through the activation ofthe adaptive immune system, agonistic CD40 antibodies can induce directtumor cell killing through inducing apoptosis of CD40-expressing solidtumor cells (Eliopoulos A G. et al., Mol Cell Biol. 2000;20(15):5503-15). The direct CD40 antibody-mediated killing of tumorcells can provide a source of tumor antigens that can be processed andpresented by APC simultaneously activated by CD40 engagement viaanti-CD40 antibodies which then can induce tumor antigen-specific Tcells, a postulated mechanism known as endogenous vaccination. Giventhat CD40 engagement can mount in an efficient anti-cancer immuneresponse, agonistic CD40 antibodies have been used successfully in avariety of preclinical tumor models, both as a single-agent and incombination with chemotherapy (Vonderheide R H and Glennie M J., ClinCancer Res. 2013; 19(5):1035-43).

To date, six CD40 mAb are under investigation in clinical trials: ChiLob 7/4 (CD40 agonistic IgG1 chimeric mAb; Cancer Research UK; ChowdhuryF. et al., Cancer Immunol Res. 2013; 2:229-40), ADC1013 (fully human,CD40 agonistic IgG1 antibody; Alligator Bioscience and Johnson &Johnson; Mangsbo S M. et al., Clin Cancer Res. 2015 Mar. 1;21(5):1115-26), APX-005 (fully humanized, CD40 agonistic IgG1 mAb;Apexigen; Bjorck P. et al. J Immunother Cancer. 2015; 3(Suppl 2): P198),SEA-CD40 (CD40 agonistic IgG1 chimeric mAb; Seattle Genetics; Gardai SJ. et al. AACR 106th Annual Meeting 2015; April 18-22, abstract 2472),as well as RO7009789 (fully human, CD40 super agonistic IgG2 mAb) areinvestigated in clinical phase I studies, and dacetuzumab (CD40 partialagonistic IgG1 chimeric mAb; Seattle Genetics; Khubchandani S. et al.,Curr Opin Investig Drugs. 2009; 10:579-87) is investigated in a clinicalphase II study. Eligible patients for these studies have solid tumors,classical Hodgkin lymphoma (HL), diffuse large B-cell lymphoma (DLBCL),or indolent lymphoma (including follicular lymphoma). Diverse activitiesranging from Fc-dependent cytotoxicity of CD40⁺ tumor cells viacomplement mediated cytotoxicity (CMC) or antibody dependent cellularcytoxicity (ADCC) to APC activation to induce anti-tumor T cellresponses as well as macrophage activation to deplete tumor and tumorstroma have been shown for these CD40 agonistic antibodies. So far thereis no conclusive explanation for this observed heterogeneity. However,recent studies indicate that this mode of action diversity can beexplained, at least in part, by differences of the anti-CD40 antibodiesin epitope specificity, isotype or Fc:FcγR interaction. For example, itappears that CD40 agonistic antibodies in vivo require crosslinkingCD40, bound by its Fab fragment on the target cell, to a Fcγ receptor,bound by its Fc fragment on a cell other than the target cell as hasbeen described for agonistic antibodies specific to otherapoptosis-inducing or immunomodulatory members of the TNFR-superfamily(Dahan R., Cancer Cell. 2016 Jun. 13; 29(6):820-31; Li F. and Ravetch J.V. Science, 2011; 333, 1030-1034; Teng M. W. et al., J. Immunol. 2009;183, 1911-1920). The proposed mechanism includes Fcγ receptor mediatedclustering of CD40 transmembrane molecules on target cells andsubsequent heightened CD40 signaling to achieve potent in vivo efficacy.

The clinical development of agonistic CD40 antibodies has providedpromising initial results. In a first clinical trial CP-870,893 hasshown clinical efficacy in patients with advanced cancer. Four out of 29patients with advanced cancer showed partial responses after receiving asingle intravenous infusion of CP-870,893 (Vonderheide R H., J ClinOncol. 2007 Mar. 1; 25(7):876-83). One out of these four patientstreated with 9 subsequent doses of CP-870,893 over one and a half yearsremained in complete remission for more than 5 years. However, the mostcommon side effects of CP-870,893 are cytokine release syndromes andthromboembolic events, so that with the dose schedules and routes ofadministration used the combined data of the phase 1 clinical studieswith more than 140 patients only indicates a limited clinical efficacyand a local administration of the antibody was suggested (Vonderheide RH, Glennie M, Clin Cancer Res. 2013, 19(5), 1035-1043). The lack ofsingle agent responses occur in part due to severe on target/off tumoreffects caused by broad CD40 expression, which results in dose limitingtoxicity (e.g. cytokine release syndrome). The development of anagonistic CD40 antibody that specifically activates APCs when CD40 iscross-linked by a tumor-specific target could reduce side effects anddecrease dose limitations, offering new therapeutic options with thepotential to generate an efficient long lasting anti-cancer immunity.

The available pre-clinical and clinical data clearly demonstrate thatthere is a high clinical need for effective agonists of CD40 that areable to induce and enhance effective endogenous immune responses tocancer. However, almost never are the effects limited to a single typeof cells or acting via a single mechanism and studies designed toelucidate inter- and intracellular signaling mechanisms have revealedincreasing levels of complexity. Known CD40 antibodies can only beadministered in relatively low doses due to dose-limiting toxicitiessuch as cytokine release syndrome and thrombocyte/endothelial cellactivation, resulting in an insufficient activation of the pathway ontarget APCs and a narrow therapeutic index. Thus, there is a need of“targeted” agonists that preferably act on a single type of cells.

The invention relates to new bispecific antigen binding moleculescapable of specific binding to CD40 and a target cell antigen. Theantigen binding molecules of the invention combine a moiety capable ofpreferred binding to tumor-specific or tumor-associated targets with amoiety capable of agonistic binding to CD40, wherein the activation ofAPCs through CD40 is provided by cross-linking through the target cellantigen, for example FAP expressed on tumor stroma cells and potentiallyalso through FAP intermediately expressed in secondary lymphoid tissues.The FAP-dependent cross-linking of the bispecific antigen bindingmolecules confines the activation of CD40-expressing cells to the tumortissue and potentially also to secondary lymphoid tissues such astumor-draining lymph nodes. In contrast to bispecific antigen bindingmolecules capable of specific binding to CD40 and to immune checkpointreceptors on activated T cells, such as CTLA-4 or PD-1, targeting to atumor target such as FAP enables CD40-mediated APC activation mainly inthe tumor stroma and tumor-draining lymph nodes where fibroblastsexpress increased levels of FAP compared to other tissues. The antigenbinding molecules of this invention may thus be able to trigger the CD40receptor not only effectively, but also very selectively at the desiredsite while overcoming the need for FcγR cross-linking thereby reducingside effects.

SUMMARY OF THE INVENTION

The present invention relates to bispecific antigen binding moleculescombining at least one moiety (antigen binding domain) capable ofspecific binding to the costimulatory TNF receptor family member CD40,with at least one antigen binding side targeting a target cell antigen.These bispecific antigen binding molecules are advantageous as they willpreferably activate costimulatory CD40 receptors at the site where thetarget cell antigen is expressed, due to their binding capabilitytowards a target cell antigen.

In one aspect, the invention provides a bispecific antigen bindingmolecule, comprising

-   (a) at least one antigen binding domain capable of specific binding    to CD40, and-   (b) at least one antigen binding domain capable of specific binding    to a target cell antigen.

In a particular aspect, the bispecific antigen binding moleculecomprises (a) at least one antigen binding domain capable of specificbinding to CD40, (b) at least one antigen binding domain capable ofspecific binding to a target cell antigen, and (c) a Fc domain composedof a first and a second subunit capable of stable association. Moreparticularly, the Fc domain composed of a first and a second subunitcapable of stable association comprises mutations that reduce effectorfunction.

In one aspect, the antigen binding domain capable of specific binding toCD40 binds to a polypeptide comprising, or consisting of, the amino acidsequence of SEQ ID NO:1.

In a further aspect, provided is a bispecific antigen binding molecule,wherein the antigen binding domain capable of specific binding to atarget cell antigen is an antigen binding domain capable of specificbinding to Fibroblast Activation Protein (FAP). In particular, theantigen binding domain capable of specific binding to FAP binds to apolypeptide comprising, or consisting of, the amino acid sequence of SEQID NO:2. Thus, in one aspect, the invention provides a bispecificantigen binding molecule, comprising (a) at least one antigen bindingdomain capable of specific binding to CD40, and (b) at least one antigenbinding domain capable of specific binding to FAP.

In one aspect, the invention provides a bispecific antigen bindingmolecule, wherein the antigen binding domain capable of specific bindingto FAP comprises

(a) a heavy chain variable region (V_(H)FAP) comprising (i) CDR-H1comprising the amino acid sequence of SEQ ID NO:3, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:4, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:5, and a light chainvariable region (V_(L)FAP) comprising (iv) CDR-L1 comprising the aminoacid sequence of SEQ ID NO:6, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO:7, and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO:8, or

-   (b) a heavy chain variable region (V_(H)FAP) comprising (i) CDR-H1    comprising the amino acid sequence of SEQ ID NO:11, (ii) CDR-H2    comprising the amino acid sequence of SEQ ID NO:12, and (iii) CDR-H3    comprising the amino acid sequence of SEQ ID NO:13, and a a light    chain variable region (V_(L)FAP) comprising (iv) CDR-L1 comprising    the amino acid sequence of SEQ ID NO:14, (v) CDR-L2 comprising the    amino acid sequence of SEQ ID NO:15, and (vi)

CDR-L3 comprising the amino acid sequence of SEQ ID NO:16.

In a further aspect, provided is a bispecific antigen binding moleculeas defined herein before, wherein the antigen binding domain capable ofspecific binding to FAP comprises

-   (a) a heavy chain variable region (V_(H)FAP) comprising an amino    acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%    identical to the amino acid sequence of SEQ ID NO:9, and a light    chain variable region (V_(L)FAP) comprising an amino acid sequence    that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to    the amino acid sequence of SEQ ID NO:10, or-   (b) a heavy chain variable region (V_(H)FAP) comprising an amino    acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%    identical to the amino acid sequence of SEQ ID NO:17, and a light    chain variable region (V_(L)FAP) comprising an amino acid sequence    that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to    the amino acid sequence of SEQ ID NO:18.

In particular, provided is a bispecific antigen binding molecule asdefined herein before, wherein the antigen binding domain capable ofspecific binding to FAP comprises (a) a heavy chain variable region(V_(H)FAP) comprising an amino acid sequence of SEQ ID NO:9, and a lightchain variable region (V_(L)FAP) comprising an amino acid sequence ofSEQ ID NO:10, or (b) a heavy chain variable region (V_(H)FAP) comprisingan amino acid sequence of SEQ ID NO:17, and a light chain variableregion (V_(L)FAP) comprising an amino acid sequence of SEQ ID NO:18.

In a further aspect, provided is a bispecific antigen binding molecule,wherein the antigen binding domain capable of specific binding to CD40comprises a heavy chain variable region (V_(H)CD40) comprising (i)CDR-H1 comprising the amino acid sequence of SEQ ID NO:19, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:20, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:21, and a light chainvariable region (V_(L)CD40) comprising (iv) CDR-L1 comprising the aminoacid sequence of SEQ ID NO:22, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO:23, and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO:24.

In another aspect, provided is a bispecific antigen binding molecule,wherein the antigen binding domain capable of specific binding to CD40binds to mouse CD40 and comprises a heavy chain variable region(V_(H)CD40) comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:27, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:28, and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:29, and a light chain variable region (V_(L)CD40) comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO:30, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:31, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:32.

Furthermore, provided is a bispecific antigen binding molecule, whereinthe antigen binding domain capable of specific binding to CD40 comprises(a) a VH comprising the amino acid sequence of SEQ ID NO:25 and a VLcomprising the amino acid sequence of SEQ ID NO:26, or (b) a VHcomprising the amino acid sequence of SEQ ID NO:33 and a VL comprisingthe amino acid sequence of SEQ ID NO:34. In a particular aspect, theinvention provides a bispecific antigen binding molecule, wherein eachof the antigen binding domains capable of specific binding to CD40comprises a VH comprising the amino acid sequence of SEQ ID NO:25 and aVL comprising the amino acid sequence of SEQ ID NO:26.

In a further aspect, provided is a bispecific antigen binding molecule,wherein the antigen binding domain capable of specific binding to CD40comprises a heavy chain variable region (V_(H)CD40) comprising

(i) CDR-H1 comprising the amino acid sequence selected from the groupconsisting of SEQ ID NO:19 and SEQ ID NO:35,

(ii) CDR-H2 comprising the amino acid sequence selected from the groupconsisting of SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38,SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 andSEQ ID NO:44, and

(iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:21, and alight chain variable region (V_(L)CD40) comprising

(iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:22,

(v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and

(vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:24.

In yet another aspect, provided is a bispecific antigen bindingmolecule, wherein the antigen binding domain capable of specific bindingto CD40 comprises a heavy chain variable region (V_(H)CD40) comprising

(i) CDR-H1 comprising the amino acid sequence selected from the groupconsisting of SEQ ID NO:19 and SEQ ID NO:261,

(ii) CDR-H2 comprising the amino acid sequence selected from the groupconsisting of

SEQ ID NO:20, SEQ ID NO:262 and SEQ ID NO:263, and

(iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:21, and alight chain variable region (V_(L)CD40) comprising

(iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:22, SEQ IDNO:264 and SEQ ID NO:265,

(v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and

(vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:24.

Furthermore, provided is a bispecific antigen binding molecule, whereinthe antigen binding domain capable of specific binding to CD40 comprises

(i) a heavy chain variable region (V_(H)CD40) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:171, SEQ IDNO:172, SEQ ID NO:173 and SEQ ID NO:174, and

(ii) a light chain variable region (V_(L)CD40) comprising the amino acidsequence selected from the group consisting of SEQ ID NO:175, SEQ IDNO:176, SEQ ID NO:177, and SEQ ID NO:178.

In particular, a bispecific antigen binding molecule is provided,wherein the antigen binding domain capable of specific binding to CD40comprises

(a) a VH comprising the amino acid sequence of SEQ ID NO:171 and a VLcomprising the amino acid sequence of SEQ ID NO:175, or

(b) a VH comprising the amino acid sequence of SEQ ID NO:173 and a VLcomprising the amino acid sequence of SEQ ID NO:177, or

(c) a VH comprising the amino acid sequence of SEQ ID NO:174 and a VLcomprising the amino acid sequence of SEQ ID NO:178, or

(d) a VH comprising the amino acid sequence of SEQ ID NO:171 and a VLcomprising the amino acid sequence of SEQ ID NO:177, or

(e) a VH comprising the amino acid sequence of SEQ ID NO:171 and a VLcomprising the amino acid sequence of SEQ ID NO:178, or

(f) a VH comprising the amino acid sequence of SEQ ID NO:173 and a VLcomprising the amino acid sequence of SEQ ID NO:175, or

(g) a VH comprising the amino acid sequence of SEQ ID NO:173 and a VLcomprising the amino acid sequence of SEQ ID NO:178, or

(h) a VH comprising the amino acid sequence of SEQ ID NO:174 and a VLcomprising the amino acid sequence of SEQ ID NO:175, or

(i) a VH comprising the amino acid sequence of SEQ ID NO:174 and a VLcomprising the amino acid sequence of SEQ ID NO:177, or

(j) a VH comprising the amino acid sequence of SEQ ID NO:171 and a VLcomprising the amino acid sequence of SEQ ID NO:176, or

(k) a VH comprising the amino acid sequence of SEQ ID NO:172 and a VLcomprising the amino acid sequence of SEQ ID NO:175, or

(l) a VH comprising the amino acid sequence of SEQ ID NO:172 and a VLcomprising the amino acid sequence of SEQ ID NO:176, or

(m) a VH comprising the amino acid sequence of SEQ ID NO:172 and a VLcomprising the amino acid sequence of SEQ ID NO:177, or

(n) a VH comprising the amino acid sequence of SEQ ID NO:172 and a VLcomprising the amino acid sequence of SEQ ID NO:178, or

(o) a VH comprising the amino acid sequence of SEQ ID NO:173 and a VLcomprising the amino acid sequence of SEQ ID NO:176, or

(p) a VH comprising the amino acid sequence of SEQ ID NO:174 and a VLcomprising the amino acid sequence of SEQ ID NO:176.

More particularly, provided is a bispecific antigen binding, wherein theantigen binding domain capable of specific binding to CD40 comprises aVH comprising the amino acid sequence of SEQ ID NO:171 and a VLcomprising the amino acid sequence of SEQ ID NO:175.

In a further aspect, provided is a bispecific antigen binding moleculeof any one of claims 1 to 7, wherein the antigen binding domain capableof specific binding to CD40 comprises

(i) a heavy chain variable region (V_(H)CD40) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:179, SEQ IDNO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183 and SEQ ID NO:184,and

(ii) a light chain variable region (V_(L)CD40) comprising the amino acidsequence selected from the group consisting of SEQ ID NO:185, SEQ IDNO:186, SEQ ID NO:187, and SEQ ID NO:188.

In particular, a bispecific antigen binding molecule is provided,wherein the antigen binding domain capable of specific binding to CD40comprises

(a) a VH comprising the amino acid sequence of SEQ ID NO:179 and a VLcomprising the amino acid sequence of SEQ ID NO:185, or

(b) a VH comprising the amino acid sequence of SEQ ID NO:180 and a VLcomprising the amino acid sequence of SEQ ID NO:185, or

(c) a VH comprising the amino acid sequence of SEQ ID NO:181 and a VLcomprising the amino acid sequence of SEQ ID NO:185, or

(d) a VH comprising the amino acid sequence of SEQ ID NO:182 and a VLcomprising the amino acid sequence of SEQ ID NO:185, or

(e) a VH comprising the amino acid sequence of SEQ ID NO:179 and a VLcomprising the amino acid sequence of SEQ ID NO:186, or

(f) a VH comprising the amino acid sequence of SEQ ID NO:180 and a VLcomprising the amino acid sequence of SEQ ID NO:186, or

(g) a VH comprising the amino acid sequence of SEQ ID NO:181 and a VLcomprising the amino acid sequence of SEQ ID NO:186, or

(h) a VH comprising the amino acid sequence of SEQ ID NO:182 and a VLcomprising the amino acid sequence of SEQ ID NO:186, or

(i) a VH comprising the amino acid sequence of SEQ ID NO:183 and a VLcomprising the amino acid sequence of SEQ ID NO:187, or

(j) a VH comprising the amino acid sequence of SEQ ID NO:183 and a VLcomprising the amino acid sequence of SEQ ID NO:188, or

(k) a VH comprising the amino acid sequence of SEQ ID NO:184 and a VLcomprising the amino acid sequence of SEQ ID NO:187, or

(l) a VH comprising the amino acid sequence of SEQ ID NO:184 and a VLcomprising the amino acid sequence of SEQ ID NO:188.

More particularly, provided is a bispecific antigen binding, wherein theantigen binding domain capable of specific binding to CD40 comprises aVH comprising the amino acid sequence of SEQ ID NO:179 and a VLcomprising the amino acid sequence of SEQ ID NO:185 or wherein theantigen binding domain capable of specific binding to CD40 comprises aVH comprising the amino acid sequence of SEQ ID NO:182 and a VLcomprising the amino acid sequence of SEQ ID NO:185.

In another aspect, provided is a bispecific antigen binding molecule,wherein the antigen binding domain capable of specific binding to CD40comprises

(i) a heavy chain variable region (V_(H)CD40) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55, and

(ii) a light chain variable region (V_(L)CD40) comprising the amino acidsequence selected from the group consisting of SEQ ID NO:56, SEQ IDNO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ IDNO:62, SEQ ID NO:63 and SEQ ID NO:64.

Particularly, a bispecific antigen binding molecule is provided, whereineach of the moieties capable of specific binding to CD40 comprises a VHcomprising the amino acid sequence of SEQ ID NO:47 and a VL comprisingthe amino acid sequence of SEQ ID NO:57.

Furthermore, provided is a bispecific antigen binding moleculecomprising

(i) at least one antigen binding domain capable of specific binding toCD40, comprising a heavy chain variable region (V_(H)CD40) comprising anamino acid sequence selected from the group consisting of SEQ ID NO:171,SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:179, SEQ IDNO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183 and SEQ ID NO:184,and a light chain variable region (V_(L)CD40) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:175, SEQ IDNO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:185, SEQ ID NO:186, SEQID NO:187 and SEQ ID NO:188, and

(ii) at least one antigen binding domain capable of specific binding toFAP, comprising a heavy chain variable region (V_(H)FAP) comprising anamino acid sequence of SEQ ID NO:9 and a light chain variable region(V_(L)FAP) comprising an amino acid sequence of SEQ ID NO:10, or a heavychain variable region (V_(H)FAP) comprising an amino acid sequence ofSEQ ID NO:17 and a light chain variable region (V_(L)FAP) comprising anamino acid sequence of SEQ ID NO:18.

In another aspect, provided is a bispecific antigen binding moleculecomprising

(i) at least one antigen binding domain capable of specific binding toCD40, comprising a heavy chain variable region (V_(H)CD40) comprising anamino acid sequence selected from the group consisting of SEQ ID NO:25,SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49,SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 andSEQ ID NO:55, and a light chain variable region (V_(L)CD40) comprisingan amino acid sequence selected from the group consisting of SEQ IDNO:26, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ IDNO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64, and

(ii) at least one antigen binding domain capable of specific binding toFAP, comprising a heavy chain variable region (V_(H)FAP) comprising anamino acid sequence of SEQ ID NO:9 and a light chain variable region(V_(L)FAP) comprising an amino acid sequence of SEQ ID NO:10, or a heavychain variable region (V_(H)FAP) comprising an amino acid sequence ofSEQ ID NO:17 and a light chain variable region (V_(L)FAP) comprising anamino acid sequence of SEQ ID NO:18.

In one aspect, the bispecific antigen binding molecule is a humanized ora chimeric antibody. In a further aspect, the bispecific antigen bindingmolecule comprises an IgG Fc region, particularly an IgG1 Fc region oran IgG4 Fc region. In particular, the Fc region comprises one or moreamino acid substitution that reduces the binding affinity of theantibody to an Fc receptor and/or effector function. In a particularaspect, provided is a bispecific antigen binding molecule, wherein theFc region is (i) of human IgG1 subclass with the amino acid mutationsL234A, L235A and P329G (numbering according to Kabat EU index), or (ii)of mouse IgG1 subclass with the amino acid mutations D265A and P329G(numbering according to Kabat EU index). Particularly, the Fc region isof human IgG1 subclass with the amino acid mutations L234A, L235A andP329G (numbering according to Kabat EU index).

In another aspect, provided is a bispecific antigen binding molecule asdefined herein before, wherein the first subunit of the Fc regioncomprises knobs and the second subunit of the Fc region comprises holesaccording to the knobs into holes method. In particular, provided is abispecific antigen binding molecule, wherein (i) the first subunit ofthe Fc region comprises the amino acid substitutions S354C and T366W(numbering according to Kabat EU index) and the second subunit of the Fcregion comprises the amino acid substitutions Y349C, T366S and Y407V(numbering according to Kabat EU index), or (ii) the first subunit ofthe Fc region comprises the amino acid substitutions K392D and K409D(numbering according to Kabat EU index) and the second subunit of the Fcregion comprises the amino acid substitutions E356K and D399K (numberingaccording to Kabat EU index). More particularly, provided is abispecific antigen binding molecule, wherein the first subunit of the Fcregion comprises the amino acid substitutions S354C and T366W (numberingaccording to Kabat EU index) and the second subunit of the Fc regioncomprises the amino acid substitutions Y349C, T366S and Y407V (numberingaccording to Kabat EU index).

In a further aspect, provided is a bispecific antigen binding molecule,wherein the bispecific antigen binding molecule comprises

(a) at least two Fab fragments capable of specific binding to CD40connected to a Fc region, and

(b) at least one antigen binding domain capable of specific binding toFAP connected to the C-terminus of the Fc region.

In another aspect, provided is a bispecific antigen binding molecule,wherein the bispecific antigen binding molecule comprises

(a) at least two Fab fragments capable of specific binding to CD40connected to a Fc region, and

(b) one antigen binding domain capable of specific binding to FAPconnected to the C-terminus of the Fc region.

In a particular aspect, the antigen binding domain capable of specificbinding to FAP connected to the C-terminus of the Fc region is across-fab fragment. Thus, provided is a bispecific antigen bindingmolecule, wherein the bispecific antigen binding molecule comprises

(a) at least two Fab fragments capable of specific binding to CD40connected to a Fc region, and

(b) a cross-fab fragment capable of specific binding to FAP connected tothe C-terminus of the Fc region.

In one aspect, provided is a bispecific antigen binding moleculecomprising

(a) two light chains and two heavy chains of an antibody comprising twoFab fragments capable of specific binding to CD40, and a Fc region, and

(b) a VH and a VL of an antigen binding domain capable specific bindingto FAP, wherein the VH is connected to the C-terminus of one of the twoheavy chains of (a), and wherein the VL is connected to the C-terminusof the other of the two heavy chains of (a).

In another aspect, provided is a bispecific antigen binding moleculecomprising

(a) two light chains and two heavy chains of an antibody comprising twoFab fragments capable of specific binding to CD40, and a Fc region, and

(b) a cross-fab fragment capable specific binding to FAP, wherein theVH-CL chain is connected to the C-terminus of one of the two heavychains of (a).

In yet another aspect, provided is a bispecific antigen binding moleculecomprising

(a) two light chains and two heavy chains of an antibody comprising twoFab fragments capable of specific binding to CD40, and a Fc region, and

(b) a cross-fab fragment capable specific binding to FAP, wherein theVL-CH1 chain is connected to the C-terminus of one of the two heavychains of (a).

Furthermore, provided is a bispecific antigen binding moleculecomprising

(a) two light chains and two heavy chains of an antibody comprising twoFab fragments capable of specific binding to CD40, and a Fc region, and

(b) two Fab fragments capable of specific binding to FAP, wherein one ofthe Fab fragments is connected to the C-terminus of one of the two heavychains of (a), and the other of the Fab fragments is connected to theC-terminus of the other of the two heavy chains of (a).

In another aspect, the invention provides a bispecific antigen bindingmolecule comprising

(a) two heavy chains, each heavy chain comprising a VH and CH1 domain ofa Fab fragment capable of specific binding to CD40 and a Fc regionsubunit,

(b) two light chains, each light chain comprising a VL and CL domain ofa Fab fragment capable of specific binding to CD40, and

(c) a VH and a VL of an antigen binding domain capable of specificbinding to FAP, wherein the VH is connected to the C-terminus of one ofthe two heavy chains of (a), and wherein the VL is connected to theC-terminus of the other of the two heavy chains of (a).

In a further aspect, provided is a bispecific antigen binding moleculecomprising

(a) two heavy chains, each heavy chain comprising a VH and CH1 domain ofa Fab fragment capable of specific binding to CD40, and a Fc regionsubunit,

(b) two light chains, each light chain comprising a VL and CL domain ofa Fab fragment capable of specific binding to CD40, and

(c) two Fab fragments capable of specific binding to FAP, wherein one ofthe Fab fragments is connected to the C-terminus of one of the two heavychains of (a), and the other of the Fab fragments is connected to theC-terminus of the other of the two heavy chains of (a).

In another aspect, provided is a bispecific antigen binding molecule,wherein the bispecific antigen binding molecule comprises

(a) two heavy chains, each heavy chain comprising a VH and CH1 domain ofa Fab fragment capable of specific binding to CD40, and a Fc regionsubunit,

(b) two light chains, each light chain comprising a VL and CL domain ofa Fab fragment capable of specific binding to CD40, and

(c) one Fab fragment capable of specific binding to FAP, wherein the Fabfragments is connected to the C-terminus of one of the two heavy chainsof (a).

In another aspect, the Fab fragment or the two Fab fragments capable ofspecific binding to FAP are crossover Fab fragments each comprising aVL-CH1 chain and a VH-CL chain, and wherein the VH-CL chain or theVL-CH1 chain is connected to the C-terminus of one of the two heavychains of (a).

In one aspect, provided is a bispecific antigen binding molecule,wherein the bispecific antigen binding molecule comprises four Fabfragments capable of specific binding to CD40. In a particular aspect,provided is a bispecific antigen binding molecule, wherein each of thetwo heavy chains of (a) as defined herein before comprises two VH-CH1chains of a Fab fragment capable of specific binding to CD40 that areconnected to each other, optionally by a peptide linker.

In another aspect, the invention provides a bispecific antigen bindingmolecule comprising

(a) two heavy chains, each heavy chain comprising two VH-CH1 chains of aFab fragment capable of specific binding to CD40 that are connected toeach other, optionally by a peptide linker, and a Fc region subunit,

(b) four light chains, each light chain comprising a VL and CL domain ofa Fab fragment capable of specific binding to CD40, and

(c) a VH and a VL of an antigen binding domain capable of specificbinding to FAP, wherein the VH is connected to the C-terminus of one ofthe two heavy chains of (a), and wherein the VL is connected to theC-terminus of the other of the two heavy chains of (a).

In another aspect, provided is a bispecific antigen binding molecule,wherein the bispecific antigen binding molecule comprises

(a) two heavy chains, each heavy chain comprising two VH-CH1 chains of aFab fragment capable of specific binding to CD40 that are connected toeach other, optionally by a peptide linker, and a Fc region subunit,

(b) four light chains, each light chain comprising a VL and CL domain ofa Fab fragment capable of specific binding to CD40, and

(c) one Fab fragment or cross-Fab fragment capable of specific bindingto FAP, wherein the Fab or cross-Fab fragment is connected to theC-terminus of one of the two heavy chains of (a).

In another aspect, provided is a bispecific antigen binding moleculecomprising

(a) two heavy chains, each heavy chain comprising two VH-CH1 chains of aFab fragment capable of specific binding to CD40 that are connected toeach other, optionally by a peptide linker, and a Fc region subunit,

(b) four light chains, each light chain comprising a VL and CL domain ofa Fab fragment capable of specific binding to CD40, and

(c) a cross-fab fragment capable specific binding to FAP, wherein theVH-CL chain of said cross-fab fragment is connected to the C-terminus ofone of the two heavy chains of (a).

In yet another aspect, provided is a bispecific antigen binding moleculecomprising

(a) two heavy chains, each heavy chain comprising two VH-CH1 chains of aFab fragment capable of specific binding to CD40 that are connected toeach other, optionally by a peptide linker, and a Fc region subunit,

(b) four light chains, each light chain comprising a VL and CL domain ofa Fab fragment capable of specific binding to CD40, and

(c) a cross-fab fragment capable specific binding to FAP, wherein theVL-CH1 chain of said cross-fab fragment is connected to the C-terminusof one of the two heavy chains of (a).

In another particular aspect, provided is a bispecific antigen bindingmolecule, wherein one or more of the Fab fragments capable of specificbinding to CD40 comprises a CL domain comprising an arginine (R) atamino acid at position 123 (numbering according to Kabat EU index) and alysine (K) at amino acid at position 124 (numbering according to KabatEU index), and a CH1 domain comprising a glutamic acid (E) at amino acidat position 147 (numbering according to Kabat EU index) and a glutamicacid (E) at amino acid at position 213 (numbering according to Kabat EUindex).

According to another aspect of the invention, there is provided anisolated polynucleotide encoding a bispecific antigen binding moleculeas described herein before. The invention further provides a vector,particularly an expression vector, comprising the isolatedpolynucleotide of the invention and a host cell comprising the isolatedpolynucleotide or the expression vector of the invention. In someaspects the host cell is a eukaryotic cell, particularly a mammaliancell.

In another aspect, provided is a method of producing a bispecificantigen binding molecule as described herein before, comprisingculturing the host cell as described above under conditions suitable forthe expression of the bispecific antigen binding molecule, and isolatingthe bispecific antigen binding molecule. The invention also encompassesthe bispecific antigen binding molecule that specifically binds to CD40and to FAP produced by the method of the invention.

The invention further provides a pharmaceutical composition comprising abispecific antigen binding molecule as described herein before and atleast one pharmaceutically acceptable excipient.

Also encompassed by the invention is the bispecific antigen bindingmolecule as described herein before, or the pharmaceutical compositioncomprising the bispecific antigen binding molecule, for use as amedicament.

In one aspect, provided is a bispecific antigen binding molecule asdescribed herein before or the pharmaceutical composition of theinvention, for use

-   (i) in inducing immune stimulation by CD40 expressing    antigen-presenting cells (APCs),-   (ii) in stimulating tumor-specific T cell response,-   (iii) in causing apoptosis of tumor cells,-   (iv) in the treatment of cancer,-   (v) in delaying progression of cancer,-   (vi) in prolonging the survival of a patient suffering from cancer,-   (vii) in the treatment of infections.

In a specific aspect, provided is the bispecific antigen bindingmolecule as described herein before or the pharmaceutical composition ofthe invention, for use in the treatment of cancer. In another specificaspect, the invention provides the bispecific antigen binding moleculeas described herein before for use in the treatment of cancer, whereinthe bispecific antigen binding molecule is administered in combinationwith a chemotherapeutic agent, radiation and/or other agents for use incancer immunotherapy. In another aspect, provided is the bispecificantigen binding molecule as described herein before or thepharmaceutical composition of the invention, for use in up-regulating orprolonging cytotoxic T cell activity.

In a further aspect, the invention provides a method of inhibiting thegrowth of tumor cells in an individual comprising administering to theindividual an effective amount of the bispecific antigen bindingmolecule as described herein before, or the pharmaceutical compositionof the invention, to inhibit the growth of the tumor cells. In anotheraspect, the invention provides a method of treating or delaying cancerin an individual comprising administering to the individual an effectiveamount of the bispecific antigen binding molecule as described hereinbefore, or the pharmaceutical composition of the invention.

Also provided is the use of the bispecific antigen binding molecule asdescribed herein before for the manufacture of a medicament for thetreatment of a disease in an individual in need thereof, in particularfor the manufacture of a medicament for the treatment of cancer, as wellas a method of treating a disease in an individual, comprisingadministering to said individual a therapeutically effective amount of acomposition comprising the bispecific antigen binding molecule of theinvention in a pharmaceutically acceptable form. In a specific aspect,the disease is cancer. In any of the above aspects the individual is amammal, particularly a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 1E, FIG. 1F show schematicrepresentations of the bispecific antigen binding molecules whichspecifically bind to human CD40 and to FAP. FIG. 1A shows a schematicrepresentation of a bispecific CD40-FAP antibody in the 4+1 formatconsisting of four CD40 binding Fab domains combined with one FAPbinding moiety with VH at the C-terminus of one heavy chain and VL atthe C-terminus of the other heavy chain (tetravalent for CD40 andmonovalent for FAP). The black point symbolizes knob-into-holemutations. FIG. 1B shows a schematic representation of a bispecificCD40-FAP antibody in the 4+2 format consisting of four CD40 binding Fabdomains combined with two FAP binding Fab domains fused each at theC-terminus of the heavy chains (tetravalent for CD40 and bivalent forFAP). FIG. 1C shows a schematic representation of a bispecific CD40-FAPantibody in the 2+1 format consisting of two CD40 binding Fab domainscombined with one FAP binding moiety with VH at the C-terminus of oneheavy chain and VL at the C-terminus of the other heavy chain (bivalentfor CD40 and monovalent for FAP). The black point symbolizesknob-into-hole mutations. FIG. 1D shows a schematic representation of abispecific CD40-FAP antibody in the 2+2 format consisting of two CD40binding Fab domains combined with two FAP binding Fab domains fused eachat the C-terminus of the heavy chains (bivalent for CD40 and bivalentfor FAP). FIG. 1E shows a schematic representation of a bispecificCD40-FAP antibody in the 2+1 format consisting of two CD40 binding Fabdomains combined with one FAP binding Fab domains fused at theC-terminus of one of the heavy chains (bivalent for CD40 and monovalentfor FAP). The black point symbolizes knob-into-hole mutations. FIG. 1Fshows a schematic representation of a bispecific CD40-FAP antibody inthe 1+1 format consisting of one CD40 binding arm combined with one FAPbinding arm (monovalent for CD40 and monovalent for FAP). The blackpoint symbolizes knob-into-hole mutations.

FIG. 2A and FIG. 2B show the binding of human tetravalent anti-CD40antibodies in a FAP-targeted monovalent or bivalent format to FAPnegative tumor cells (FIG. 2A) and FAP positive tumor cells (FIG. 2B).The transgenic modified mouse embryonic fibroblast NIH/3T3-huFAP clone19 expresses high levels of human fibroblast activation protein (huFAP),whereas the parental cell line NIH/3T3-wt expresses no huFAP. Only thetetravalent anti-CD40 antigen binding molecules with either one or twoFAP binding moieties but not the non-FAP targeted formats efficientlybind to NIH/3T3-huFAP cells (FIG. 2B). The bivalent FAP construct bindsstronger than the monovalent construct. In contrast, no binding of theFAP-targeted anti-CD40 antibodies to the NIH/3T3-wt cells was detected(FIG. 2A). Shown is the binding as median of fluorescence intensity(MFI) of phycoerythrin (PE)-labeled anti-human IgG Fcγ-specific goat IgGF(ab′)2 fragment which is used as secondary detection antibody. MFI wasmeasured by flow cytometry. The x-axis shows the concentration ofantibody constructs.

FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F, FIG. 3G, FIG. 3Hshow the in vitro activation of human B cells by mono- or bivalentFAP-targeted anti-CD40 constructs. With NIH/3T3-FAP cells the bispecificantibody monovalent for FAP induced a similar increase of B cellactivation marker expression (CD70, CD80, CD83, and CD86) as thebivalent FAP-targeted molecule. Moreover, the B cell activation markerupregulation by FAP-targeted bispecific antigen-binding molecules wascomparable to the upregulation induced by the FAP-independent positivecontrol antibodies. In the absence of FAP (NIH/3T3-wt cells) no increaseof B cell activation markers could be observed with the bispecificantigen binding molecules, while positive control antibodies induced anupregulation of activation marker. Shown is the percentage of CD70 (FIG.3A and FIG. 3B), CD80 (FIG. 3C and FIG. 3D), CD83 (FIG. 3E and FIG. 3F)and CD86 (FIG. 3G and FIG. 3H) positive vital B cells after 2 daysincubation with the indicated titrated antibodies. The x-axis shows theconcentration of antibody constructs. The effect on NIH/3T3-FAP cells isshown In FIG. 3A, FIG. 3C, FIG. 3E and FIG. 3G, respectively, while theeffect on NIH/3T3-wt cells is shown in FIG. 3B, FIG. 3D, FIG. 3F andFIG. 3H, respectively.

FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F and FIG. 5A, FIG.5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 5G, FIG. 5H show the invitro activation of human B cells by mono- or bivalent FAP-targetedhuman anti-CD40 constructs in the presence of FAP-coated or uncoatedDynabeads® after 2 days (FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E,FIG. 4F) or 5 days incubation (FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG.5E, FIG. 5F, FIG. 5G, FIG. 5H). After 2 days incubation with FAP-coatedbeads the bispecific antibodies monovalent for FAP induced a similarincrease of B cell activation marker expression (CD70, CD83, and CD86)as the bivalent FAP-targeted molecules. Moreover, the B cell activationmarker upregulation by FAP-targeted bispecific antigen-binding moleculeswas comparable to the upregulation induced by the FAP-independentpositive control antibodies. In the absence of FAP (uncoated beads) noincrease of B cell activation markers could be observed with thebispecific antigen binding molecules, while positive control antibodiesinduced an upregulation of activation markers. After 5 days B cellincubation with FAP-coated Dynabeads® FAP-targeted human anti-CD40constructs induced a FAP-dependent upregulation of CD80 and CD86expression on B cells. Compared to the FAP-independent upregulation ofCD86 induced by RO7009789 or cross-linked SGN-40, CD86 upregulationinduced by FAP-dependent bispecific antigen binding molecules wasslightly lower. For CD70 and CD83 no or only very limited upregulationcould be observed with the bispecifc antibodies targeting FAP and CD40,while the positive control antibodies clearly showed an effect on theseB cell activation markers. Shown is the percentage of CD70 (FIG. 4A,FIG. 4B, FIG. 5A and FIG. 5B, respectively), CD80 (FIG. 5C and FIG. 5D,respectively), CD83 (FIG. 4C, FIG. 4D, FIG. 5E and FIG. 5F,respectively) and CD86 (FIG. 4E, FIG. 4F, FIG. 5G and FIG. 5H,respectively) positive vital B cells after 2 days or 5 days incubationwith the indicated titrated antibodies. The x-axis shows theconcentration of antibody constructs.

FIG. 6A and FIG. 6B show the IL-6 secretion of human B cells treatedwith different FAP-targeted and untargeted agonistic anti-CD40antibodies in the presence of FAP-coated (FIG. 6A) or uncoated beads(FIG. 6B) after 5 days incubation. In the presence of FAP the monovalentas well as the bivalent FAP-targeted anti-CD40 antibody induced asimilar increased IL-6 secretion as compared to the FAP-independentpositive control antibodies RO7009789 and SGN40. In contrast, B cellstreated with the untargeted negative control antibodies expressedsimilar low IL-6 levels as untreated B cells. In the absence of FAP(uncoated beads) no increase in IL-6 production was detected with thebispecific antigen binding molecules. Shown is the IL-6 amount in thesupernatant of human B cells cultured for five days with the indicatedtitrated antibodies measured by ELISA. The x-axis shows theconcentration of antibody constructs.

FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG. 7F, FIG. 7G, FIG. 7Hshow the in vitro activation of human monocyte-derived DCs (moDCs) bymono- or bivalent FAP-targeted human anti-CD40 constructs in thepresence of FAP-coated or uncoated Dynabeads® after 2 days incubation.In the presence of FAP-coated beads the bispecific antibody monovalentfor FAP induced a similar increase of DC activation marker expression(CD70, CD80, and CD83) as the bivalent FAP-targeted molecule, whereas inthe absence of FAP (uncoated beads) no activation marker upregulationwas detected. Moreover, the upregulation of CD80 and CD83 byFAP-targeted bispecific antigen-binding molecules was comparable to theupregulation induced by the FAP-independent positive control antibodies.A higher upregulation of CD70 on DCs was observed for RO709789 comparedto all other tested antibodies. In contrast, CD86 expression was notsignificantly changed on DCs incubated with the different anti-CD40antibodies compared to untreated DCs. Shown is the percentage of CD70(FIG. 7A and FIG. 7B), CD80 (FIG. 7C and FIG. 7D), CD83 (FIG. 7E andFIG. 7F) and CD86 (FIG. 7G and FIG. 7H) positive vital moDCs after 2days incubation with the indicated titrated antibodies. The x-axis showsthe concentration of antibody constructs.

FIG. 8A and FIG. 8B show the in vitro activation of HEK-Blue™ CD40Lcells by mono- or bivalent FAP-targeted human anti-CD40 constructs inthe presence of FAP-coated or uncoated Dynabeads® after 8 hoursincubation. In the presence of FAP-coated beads the bispecific antibodymonovalent for FAP and bivalent for CD40 induced a similar increase ofSEAP production as the bispecific antibody bivalent for FAP and CD40,whereas in the absence of FAP (uncoated beads) no SEAP production wasdetected. Moreover, an upregulation of SEAP production by FAP-targetedantibodies tetravalent for CD40 was observed in the presence of FAP.However, SEAP production was also observed in the absence of FAP in thesupernatant of reporter cells treated with FAP-targeted antibodiestetravalent for CD40. The negative control antibodies tetravalent forhuman CD40 with one or two DP47 domains instead of a FAP binding domaininduced comparable SEAP production in HEK-Blue™ CD40L cells in thepresence and absence of FAP and the positive control antibodySGN-40+F(ab) induced similar levels of SEAP production as compared toFAP-targeted bispecific antibodies bivalent or tetravalent for humanCD40 in the presence of FAP-coated beads. Shown is the absorption at awavelength of 650 nm which correlates with the amount of hydrolyzedsubstrate by SEAP. The x-axis shows the concentration of antibodyconstructs.

FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, FIG. 9F, FIG. 9G, FIG. 9Hshow the T cell priming of SIINFEKL-pulsed DCs activated by FAP-targetedanti-CD40 binding molecules. DCs isolated from huCD40 transgenic mice(similar expression pattern of human CD40 and mouse CD40), pulsed withlow amounts of SIINFEKL and stimulated with FAP-dependent bispecificanti-CD40 antibodies as well as FAP-coated beads induced a strongproliferation of antigen-specific T cells. In contrast, in the absenceof FAP (uncoated beads) no T cell proliferation was induced by DCsstimulated with FAP-targeted anti-CD40 antibodies. T cell proliferationlevels induced by DCs stimulated with the murine or the human bispecificantigen binding molecules with four CD40 and two FAP binding moietieswas comparable. No significant upregulation of the T cell activationmarkers CD44 and CD25 or IFNγ production was observed for T cellsco-cultured with DCs pre-stimulated with different agonistic anti-CD40antibodies. Only DCs pulsed with high amounts of SIINFEKL induced aclear expression increase of these markers compared to the untreatedcondition. Shown is the percentage of proliferating (CFSE-low), IFNγ,CD25, and CD44 positive vital CFSE-labeled murine CD3⁺CD8⁺ OT-1 T cellsco-cultured with huCD40 tg DCs pre-incubated with the indicated titratedantibodies. The x-axis shows the concentration of antibody constructs.

FIG. 9I and FIG. 9J show the concentration of IL-2 and IL-12(p40)measured in the supernatant of T cell primed by SIINFEKL-pulsedFAP-targeted anti-CD40 antibody-activated DCs. In the co-culture of OT-1T cells and huCD40 tg DCs pulsed with low amounts of SIINFEKL andstimulated with FAP-dependent bispecific anti-CD40 antibodies as well asFAP-coated beads increased IL-2 and IL-12(p40) levels were detectedcompared to OT-1 T cells co-cultured with huCD40 tg DCs pre-stimulatedwith FAP-targeted antibodies in the absence of FAP. Moreover, the murinebivalent FAP-targeted anti-CD40 antibody induced a markedly highersecretion of IL-12(p40) as the human equivalent bispecific antigenbinding molecule. IL-2 secretion was increased to a similar extent withboth, the anti-human CD40 and the anti-mouse CD40 bispecific antigenbinding molecules in a FAP-dependent way. Shown is the IL-2 andIL-12(p40) amount in the supernatant of murine CD3⁺CD8⁺ OT-1 T cellsco-cultured with huCD40 tg DCs pre-incubated with the indicated titratedantibodies measured by ELISA. The x-axis shows the concentration ofantibody constructs.

FIG. 10A, FIG. 10B, FIG. 10C, FIG. 11A, FIG. 11B, FIG. 11C, FIG. 12A,FIG. 12B, FIG. 12C show the T cell priming of OVA-pulsed DCs activatedby FAP-targeted anti-CD40 binding molecules. DCs isolated from huCD40transgenic mice, treated with DEC205-OVA conjugate and stimulated withFAP-dependent bispecific anti-CD40 antibodies as well as FAP-coatedbeads induced a strong proliferation and CD25 as well as CD44 expressionof antigen-specific T cells. In contrast, in the absence of FAP(uncoated beads) or OVA (DEC only) no T cell proliferation andactivation was induced by DCs stimulated with FAP-targeted anti-CD40antibodies. T cell proliferation and CD25 as well as CD44 expressionlevels induced by DCs stimulated with the murine or the human bispecificantigen binding molecules with four CD40 and two FAP binding moietieswas comparable. DCs pulsed with high amounts of SIINFEKL instead ofDEC205-OVA conjugate also induced a strong T cell proliferation andexpression of the activation markers CD25 and CD44. Shown is thepercentage of proliferating (CFSE-low) (FIG. 10A, FIG. 10B, FIG. 10C),CD25 (FIG. 11A, FIG. 11B, FIG. 11C) and CD44 (FIG. 12A, FIG. 12B, FIG.12C) positive vital CFSE-labeled murine CD3⁺CD8⁺ OT-1 T cellsco-cultured with huCD40 tg DCs pre-incubated with the indicated titratedantibodies in the presence or absence of OVA. The x-axis shows theconcentration of antibody constructs.

FIG. 13A, FIG. 13B, FIG. 13C show IFNγ levels measured in thesupernatants of T cells co-cultured with OVA-pulsed DCs activated byFAP-targeted anti-CD40 binding molecules. IFNγ levels were elevated inconditions with T cells co-cultured with DCs treated with the anti-humanCD40 FAP-targeting antibody in the presence of FAP (FIG. 13A). Inaddition, IFNy secretion was increased to a similar extent with both,the anti-human CD40 and the anti-mouse CD40 bispecific antigen bindingmolecules in a FAP-dependent way. Shown is the IFNγ amount in thesupernatant of murine CD3⁺CD8⁺ OT-1 T cells co-cultured with huCD40 tgDCs pre-incubated with the indicated titrated antibodies measured byELISA. The x-axis shows the concentration of antibody constructs.

FIG. 14A, FIG. 14B, FIG. 14C, FIG. 14D, FIG. 14E, FIG. 14F, FIG. 14G,FIG. 14H show the in vitro activation of murine B cells by mono- orbivalent FAP-targeted mouse anti-CD40 constructs in the presence ofFAP-coated or uncoated Dynabeads® after 2 days incubation. WithFAP-coated beads the bispecific antibody monovalent for FAP induced asimilar increase of B cell activation marker expression (CD70, CD80, andCD86) as the bivalent FAP-targeted molecule. Moreover, a significant Bcell activation marker upregulation was also observed for B cellstreated with the FAP-independent positive control antibody FGK4.5. Inthe absence of FAP (uncoated beads) no increase of the B cell activationmarkers CD70 and CD80 could be observed with the bispecific antigenbinding molecules. In contrast, the positive control antibody induced anupregulation of CD70 and CD80 irrespective of FAP pre-treatment. WhileCD86 upregulation was FAP-dependent with the tetravalent anti-mouse CD40antibody possessing two FAP binding moieties, a FAP-independent effectwas observed for the bispecific antigen binding molecule having only oneFAP binding site. In addition, a FAP-independent upregulation of MHC-IIexpression was observed for all tested bispecific antigen bindingmolecules. Shown is the percentage of CD70 (FIG. 14A and FIG. 14B), CD80(FIG. 14C and FIG. 14D), CD86 (FIG. 14E and FIG. 14F) and MHCII (FIG.14G and FIG. 14H) positive vital B cells after 2 days incubation withthe indicated titrated antibodies. The x-axis shows the concentration ofantibody constructs.

FIG. 15A, FIG. 15B, FIG. 15C, FIG. 15D, FIG. 15E, FIG. 15F, FIG. 15Gshow schematic representations of the bispecific antigen bindingmolecules which specifically bind to human CD40 and to FAP or DP47. FIG.15A shows a schematic representation of a bispecific CD40-DP47 antibodyin the 4+1 format consisting of four CD40 binding Fab domains combinedwith one DP47 binding moiety with VH at the C-terminus of one heavychain and VL at the C-terminus of the other heavy chain (tetravalent forCD40 and monovalent for DP47). The black point symbolizes knob-into-holemutations. FIG. 15B shows a schematic representation of a bispecificCD40-DP47 antibody in the 4+2 format consisting of four CD40 binding Fabdomains combined with two DP47 binding Fab domains fused each at theC-terminus of the heavy chains (tetravalent for CD40 and bivalent forDP47). FIG. 15C shows a schematic representation of a bispecificCD40-FAP antibody in the 1+1 format consisting of one CD40 binding armcombined with one FAP binding arm (monovalent for CD40 and monovalentfor FAP). FIG. 15D shows a schematic scheme of an exemplary bispecificCD40-FAP antibody in the 2+1 format consisting of two CD40 binding Fabdomains combined with one FAP binding Fab domain as part of one of thetwo CD40 binding arms. FIG. 15E shows a schematic representation of abispecific CD40-FAP antibody in the 4+1 format consisting of four CD40binding Fab domains combined with one FAP binding Fab domains fused atthe C-terminus of one of the heavy chains (tetravalent for CD40 andmonovalent for FAP). FIG. 15F shows a schematic representation of abispecific CD40-FAP antibody in the 4+1 format consisting of four CD40binding Fab domains combined with one FAP binding Fab domains fused atthe C-terminus of one of the heavy chains. The VH2a and VL2a CD40binding domains were obtained from an in-house humanization of themurine S2C6 CD40 binding domain. FIG. 15G shows a schematicrepresentation of a bispecific CD40-FAP antibody in the 4+1 formatconsisting of four CD40 binding Fab domains combined with one FAPbinding Fab domains fused at the C-terminus of one of the heavy chains.The VH2d and VL2a CD40 binding domains were obtained from an in-househumanization of the murine S2C6 CD40 binding domain. FIG. 15H shows aschematic representation of a bispecific CD40-FAP antibodies in the 2+1format consisting of two CD40 binding moieties combined with one FAPbinding moiety as crossover fab fragment, wherein the VL-CH1 chain isfused at the C-terminus of the Fc knob chain. FIG. 15I shows a schematicrepresentation of a bispecific CD40-FAP antibodies in the 2+1 formatconsisting of two CD40 binding moieties combined with one FAP bindingmoiety as crossover fab fragment, wherein the VH-CL chain is fused atthe C-terminus of the Fc knob chain.

FIG. 16A shows the parental murine FGK4.5 antibody (P1AD3449). FIG. 16B,FIG. 16C, FIG. 16D, FIG. 16E show schematic representations of thebispecific antigen binding molecules which specifically bind to mouseCD40 and to FAP or DP47. FIG. 16B shows a schematic representation of abispecific CD40-FAP antibody in the 4+1 format consisting of four mouseCD40 binding Fab domains combined with one FAP binding moiety with VH atthe C-terminus of one heavy chain and VL at the C-terminus of the otherheavy chain (tetravalent for CD40 and monovalent for FAP). The blackpoint symbolizes DD/KK mutations in the Fc and binding to Fc receptorsis inhibited by D270A/P329G mutations. FIG. 16C shows a schematicrepresentation of a bispecific CD40-FAP antibody in the 4+2 formatconsisting of four mouse CD40 binding Fab domains combined with two FAPbinding Fab domains fused each at the C-terminus of the heavy chains(tetravalent for CD40 and bivalent for FAP). Binding to Fc receptors isinhibited by D270A/P329G mutations. FIG. 16D shows a schematicrepresentation of a bispecific CD40-DP47 antibody in the 4+1 formatconsisting of four mouse CD40 binding Fab domains combined with one DP47binding moiety with VH at the C-terminus of one heavy chain and VL atthe C-terminus of the other heavy chain (tetravalent for CD40 andmonovalent for DP47). The black point symbolizes DD/KK mutations in theFc and binding to Fc receptors is inhibited by D270A/P329G mutations.FIG. 16E shows a schematic representation of a bispecific CD40-DP47antibody in the 4+2 format consisting of four mouse CD40 binding Fabdomains combined with two DP47 binding Fab domains fused each at theC-terminus of the heavy chains (tetravalent for CD40 and bivalent forDP47). Binding to Fc receptors is inhibited by D270A/P329G mutations.

FIG. 17 shows the binding of human tetravalent, bivalent or monovalentanti-CD40 antibodies in a FAP-targeted monovalent or bivalent format toFAP positive tumor cells. The transgenic modified mouse embryonicfibroblast NIH/3T3-mFAP cell line expresses high levels of murinefibroblast activation protein (mFAP). All depicted constructs vary intheir binding strength (EC₅₀ values as well as signal strength) toNIH/3T3-mFAP cells. Only the anti-CD40 antigen binding molecules witheither one or two FAP binding moieties but not the non-FAP-targetedformats (P1AD4574 and P1AD4465) efficiently bind to NIH/3T3-mFAP cells.The bivalent FAP constructs with C-terminal FAP binding domains bindstronger than the monovalent construct with C-terminal FAP bindingdomains. The strongest FAP binding was observed for the 1+1 format.Shown is the binding as median of fluorescence intensity (MFI) ofphycoerythrin (PE)-labeled anti-human IgG Fcγ-specific goat IgG F(ab′)2fragment which is used as secondary detection antibody. MFI was measuredby flow cytometry. The x-axis shows the concentration of antibodyconstructs.

FIG. 18 shows the binding of human tetravalent, bivalent or monovalentanti-CD40 antibodies in a FAP-targeted monovalent or bivalent format toDaudi cells, a B lymphoblast cell line with high surface expressionlevels of human CD40. All depicted constructs bind to CD40 but vary intheir binding strength (EC₅₀ values as well as signal strength) toCD40-positive Daudi cells. Bivalent anti-CD40 antibodies show higherEC₅₀ levels and reach higher binding plateaus compared to tetravalentanti-CD40 antibodies. The highest EC₅₀ value combined with the lowestbinding plateau was observed for the 1+1 format. Binding of anti-CD40antibodies to cell surface proteins was detected with an anti-human IgGFcγ-specific goat IgG F(ab′)2 fragment conjugated to phycoerythrin (PE)using FACS analysis. MFI was measured by flow cytometry and baselinecorrected by subtracting the MFI of the blank control. The x-axis showsthe concentration of antibody constructs.

FIG. 19A and FIG. 19B show the in vitro activation of HEK-Blue™ CD40Lcells by mono- or bivalent FAP-targeted human anti-CD40 constructs inthe presence of FAP-coated (FIG. 19A) or uncoated Dynabeads® (FIG. 19B)after 24 hours incubation. In the presence of FAP-coated beads thebispecific antibody monovalent for FAP and mono- or bivalent for CD40induced a similar increase of SEAP production as the bispecific antibodybivalent for FAP and CD40, whereas in the absence of FAP (uncoatedbeads) no or low SEAP production was detected. Moreover, an upregulationof SEAP production by FAP-targeted antibodies tetravalent for CD40 wasobserved in the presence of FAP. However, SEAP production was alsoobserved in the absence of FAP in the supernatant of reporter cellstreated with FAP-targeted antibodies tetravalent for CD40. The negativecontrol antibody tetravalent for human CD40 with one DP47 domain insteadof a FAP binding domain induced comparable SEAP production in HEK-Blue™CD40L cells in the presence and absence of FAP and the positive controlantibody P1AD4470+F(ab) induced similar levels of SEAP production ascompared to FAP-targeted bispecific antibodies bivalent or tetravalentfor human CD40 in the presence of FAP-coated beads. Shown is theabsorption at a wavelength of 650 nm which correlates with the amount ofhydrolyzed substrate by SEAP. The x-axis shows the concentration ofantibody constructs. The EC₅₀ values of HEK-Blue™ CD40L cell activationin the presence of FAP-coated beads are summarized in Table 11. The EC₅₀values of all tested antibodies tetravalent for CD40 were comparable andlower compared to the EC₅₀ values of the depicted antibodies bivalentfor CD40. The highest EC₅₀ value was detected for the 1+1 format.

FIG. 20A and FIG. 20B show the in vitro activation of human Daudi cellsby mono- or bivalent FAP-targeted human anti-CD40 constructs in thepresence of FAP-coated (FIG. 20A) or uncoated Dynabeads® (FIG. 20B)after 2 days incubation. With FAP-coated beads the bispecific antibodiesmonovalent for FAP induced a similar increase of the B cell activationmarker expression CD70 as the bivalent FAP-targeted molecules. Moreover,the B cell activation marker upregulation by FAP-targeted bispecificantigen-binding molecules was higher comparable to the upregulationinduced by the FAP-independent positive control antibodies. In theabsence of FAP (uncoated beads) no increase of CD70 could be observedwith the depicted FAP-targeted bispecific antibodies mono- or bivalentfor CD40, while tetravalent CD40 binding molecules induce anupregulation of CD70, but to a lesser extent than in the presence ofFAP. Shown is the percentage of CD70 positive vital Daudi cells after 2days incubation with the indicated titrated antibodies. The x-axis showsthe concentration of antibody constructs. The EC₅₀ values of activationin the presence of FAP-coated beads are summarized in Table 9. The EC₅₀values of all FAP-targeted antibodies tetravalent for CD40 werecomparable and lower compared to the EC₅₀ values of the depictedantibodies bivalent for CD40. The highest EC₅₀ values were detected forthe positive control antibody P1AD4470 and the 1+1 format.

FIG. 21A and FIG. 21B show the in vitro activation of human B cells bymono- or bivalent FAP-targeted human anti-CD40 constructs in thepresence of FAP-coated (FIG. 21A) or uncoated Dynabeads® (FIG. 21B)after 2 days incubation. With FAP-coated beads the bispecific antibodiesmonovalent for FAP induced a similar increase of the B cell activationmarker expression CD86 as the bivalent FAP-targeted molecules. Comparedto the FAP-independent upregulation of CD86 induced by cross-linked CD40antibody (P1AD4470), CD86 upregulation induced by FAP-dependentbispecific antigen binding molecules was slightly lower. In the absenceof FAP (uncoated beads) no increase of CD86 expression could be observedwith the bispecific antigen binding molecules, while positive controlantibodies induced an upregulation of activation markers. Shown is thepercentage of CD86 positive vital B cells after 2 days incubation withthe indicated titrated antibodies. The x-axis shows the concentration ofantibody constructs. The EC₅₀ values of activation in the presence ofFAP-coated beads are summarized in Table 10. The EC₅₀ values of allFAP-targeted antibodies tetravalent for CD40 were comparable and lowercompared to the EC₅₀ values of the depicted FAP-targeted antibodiesbivalent for CD40. The highest EC₅₀ values were detected for the 2+1,2+2, and 1+1 format.

FIG. 22A and FIG. 22B show the T cell priming of OVA-pulsed DCsactivated by FAP-targeted anti-CD40 binding molecules in the presence(FIG. 22A) or absence (FIG. 22B) of FAP. DCs isolated from huCD40transgenic mice, treated with DEC205-OVA conjugate and stimulated withFAP-dependent bispecific anti-CD40 antibodies as well as FAP-coatedbeads induced a strong proliferation of antigen-specific T cells. Incontrast, in the absence of FAP (uncoated beads) no or little T cellproliferation and activation was induced by DCs stimulated withFAP-targeted anti-CD40 antibodies. T cell proliferation induced by DCsstimulated with the human bispecific antigen binding molecules with one,two or four CD40 and one or two FAP binding moieties was comparable. DCspulsed with high amounts of SIINFEKL instead of DEC205-OVA conjugateinduced a strong T cell proliferation. Shown is the percentage ofproliferating (CFSE-low) vital CFSE-labeled murine CD3⁺CD8⁺ OT-1 T cellsco-cultured with huCD40 tg DCs pre-incubated with the indicated titratedantibodies in the presence of OVA (FIG. 22A and FIG. 22B). The x-axisshows the concentration of antibody constructs.

FIG. 23A, FIG. 23B, FIG. 23C, FIG. 23D, and FIG. 23E; and FIG. 24A, FIG.24B, FIG. 24C, and FIG. 24D show enzyme serum levels, body weight,spleen weight, DC activation and T cell proliferation in mice injectedwith a FAP-expressing murine colon adenocarcinoma tumor cell line(MC38-FAP) and treated with either FGK4.5 (PlAD3449) or FGK4.5×FAP 4+1(P1AD4520) or vehicle alone. In contrast to mice treated withnon-targeted CD40 mAb (FGK4.5), treatment with FAP-CD40 (FGK4.5) 4+1,(i.e a bispecific antibody tetravalent for CD40 and monovalent for FAP)did not induce liver injury as no increase in serum enzymes indicativeof liver injury was observed. This is shown for 3 animals per group inFIG. 23A for alanine aminotransferase (ALT), in FIG. 23B for glutamatedehydrogenase (GDH) and in FIG. 23C for sorbitol dehydrogenase (SDH).Moreover, no decrease in body weight (FIG. 23D) and less increase inspleen weight (FIG. 23E) was observed in mice treated with FGK4.5×FAP(P1AD4520) compared to mice treated with the parental untargeted CD40antibody (P1AD3449). DC activation in the tumor-draining lymph nodesthree days post therapy injection (FIG. 24A and FIG. 24B) and T cellproliferation in tumor eight days post therapy injection (FIG. 24C andFIG. 24D) was significantly increased in FGK4.5- and FGK4.5×FAP4+1-treated animals compared to vehicle-treated animals. In FIG. 23A,FIG. 23B, and FIG. 23C the y-axis shows serum enzyme levels in units perliter and the x-axis shows individual mice treated with FGK4.5,FGK4.5×FAP 4+1 or vehicle alone. In FIG. 23D the y-axis shows the bodyweight in gram of mice treated FGK4.5, FGK4.5×FAP 4+1 or vehicle aloneand the x-axis shows the days post tumor injection. FIG. 23E shows thespleen weight of mice treated with FGK4.5, FGK4.5×FAP 4+1 or vehiclealone three days post therapy injection. FIG. 24A, FIG. 24B, FIG. 24C,and FIG. 24D show the CD86 (FIG. 24A) and CD70 expression (FIG. 24B) ofDCs in the tumor-draining lymph node and the Ki67 expression of CD8⁺ Tcells (FIG. 24C) and the total numbers of CD8⁺ T cell (FIG. 24D) in thetumor three and eight days post therapy injection, respectively.((*p<0.05, **p<0.01, ***p<0.001, unpaired, two-tailed Student's test).

FIG. 25A and FIG. 25B show the in vitro activation of human B cells bybivalent FAP-targeted human anti-CD40 constructs in the presence ofFAP-coated (FIG. 25A and FIG. 25C) or uncoated Dynabeads® (FIG. 25B andFIG. 25D) after 2 days incubation. Compared to the FAP-independentupregulation of CD70 and CD86 induced by the cross-linked CD40 antibody(P1AA5145), CD70 upregulation (FIG. 25A) and CD86 upregulation (FIG.25C) induced by FAP-dependent bispecific antigen binding molecules wasslightly lower. In the absence of FAP (uncoated beads) no increase ofCD70 (FIG. 25B) or CD86 expression (FIG. 25D) could be observed with thebispecific antigen binding molecules, while positive control antibodiesinduced an upregulation of activation markers. Shown is the percentageof CD70 or CD86 positive vital B cells after 2 days incubation with theindicated titrated antibodies. The x-axis shows the concentration ofantibody constructs.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as generally used in the art to which thisinvention belongs. For purposes of interpreting this specification, thefollowing definitions will apply and whenever appropriate, terms used inthe singular will also include the plural and vice versa.

As used herein, the term “antigen binding molecule” refers in itsbroadest sense to a molecule that specifically binds an antigenicdeterminant. Examples of antigen binding molecules are antibodies,antibody fragments and scaffold antigen binding proteins.

As used herein, the term “antigen binding domain capable of specificbinding to a target cell antigen” or “moiety capable of specific bindingto a target cell antigen” refers to a polypeptide molecule thatspecifically binds to an antigenic determinant. In one aspect, theantigen binding domain is able to activate signaling through its targetcell antigen. In a particular aspect, the antigen binding domain is ableto direct the entity to which it is attached (e.g. the CD40 agonist) toa target site, for example to a specific type of tumor cell or tumorstroma bearing the antigenic determinant. Antigen binding domainscapable of specific binding to a target cell antigen include antibodiesand fragments thereof as further defined herein. In addition, antigenbinding domains capable of specific binding to a target cell antigeninclude scaffold antigen binding proteins as further defined herein,e.g. binding domains which are based on designed repeat proteins ordesigned repeat domains (see e.g. WO 2002/020565).

In relation to an antibody or fragment thereof, the term “antigenbinding domain capable of specific binding to a target cell antigen”refers to the part of the molecule that comprises the area whichspecifically binds to and is complementary to part or all of an antigen.A antigen binding domain capable of specific antigen binding may beprovided, for example, by one or more antibody variable domains (alsocalled antibody variable regions). Particularly, an antigen bindingdomain capable of specific antigen binding comprises an antibody lightchain variable region (VL) and an antibody heavy chain variable region(VH). In another aspect, the “antigen binding domain capable of specificbinding to a target cell antigen ” can also be a Fab fragment or across-Fab fragment.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, monospecific and multispecificantibodies (e.g., bispecific antibodies), and antibody fragments so longas they exhibit the desired antigen-binding activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g. containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen.

The term “monospecific” antibody as used herein denotes an antibody thathas one or more binding sites each of which bind to the same epitope ofthe same antigen. The term “bispecific” means that the antigen bindingmolecule is able to specifically bind to at least two distinct antigenicdeterminants. Typically, a bispecific antigen binding molecule comprisestwo antigen binding sites, each of which is specific for a differentantigenic determinant. In certain embodiments the bispecific antigenbinding molecule is capable of simultaneously binding two antigenicdeterminants, particularly two antigenic determinants expressed on twodistinct cells. A bispecific antigen binding molecule as describedherein can also form part of a multispecific antibody.

The term “valent” as used within the current application denotes thepresence of a specified number of binding sites specific for onedistinct antigenic determinant in an antigen binding molecule that arespecific for one distinct antigenic determinant. As such, the terms“bivalent”, “tetravalent”, and “hexavalent” denote the presence of twobinding sites, four binding sites, and six binding sites specific for acertain antigenic determinant, respectively, in an antigen bindingmolecule. In particular aspects of the invention, the bispecific antigenbinding molecules according to the invention can be monovalent for acertain antigenic determinant, meaning that they have only one bindingsite for said antigenic determinant or they can be bivalent ortetravalent for a certain antigenic determinant, meaning that they havetwo binding sites or four binding sites, respectively, for saidantigenic determinant.

The terms “full length antibody”, “intact antibody”, and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure.“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG-classantibodies are heterotetrameric glycoproteins of about 150,000 daltons,composed of two light chains and two heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3),also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable region (VL), also called avariable light domain or a light chain variable domain, followed by alight chain constant domain (CL), also called a light chain constantregion. The heavy chain of an antibody may be assigned to one of fivetypes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ1 (IgG1), γ2 (IgG2),γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies, triabodies, tetrabodies, cross-Fab fragments; linearantibodies; single-chain antibody molecules (e.g. scFv); and singledomain antibodies. For a review of certain antibody fragments, seeHudson et al., Nat Med 9, 129-134 (2003). For a review of scFvfragments, see e.g. Plückthun, in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos.5,571,894 and 5,587,458. For discussion of Fab and F(ab′)2 fragmentscomprising salvage receptor binding epitope residues and havingincreased in vivo half-life, see U.S. Pat. No. 5,869,046. Diabodies areantibody fragments with two antigen-binding sites that may be bivalentor bispecific, see, for example, EP 404,097; WO 1993/01161; Hudson etal., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad SciUSA 90, 6444-6448 (1993). Triabodies and tetrabodies are also describedin Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodiesare antibody fragments comprising all or a portion of the heavy chainvariable domain or all or a portion of the light chain variable domainof an antibody. In certain embodiments, a single-domain antibody is ahuman single-domain antibody (Domantis, Inc., Waltham, Mass.; see e.g.U.S. Pat. No. 6,248,516 B1). Antibody fragments can be made by varioustechniques, including but not limited to proteolytic digestion of anintact antibody as well as production by recombinant host cells (e.g. E.coli or phage), as described herein.

Papain digestion of intact antibodies produces two identicalantigen-binding fragments, called “Fab” fragments containing each theheavy- and light-chain variable domains and also the constant domain ofthe light chain and the first constant domain (CH1) of the heavy chain.As used herein, Thus, the term “Fab fragment” refers to an antibodyfragment comprising a light chain fragment comprising a VL domain and aconstant domain of a light chain (CL), and a VH domain and a firstconstant domain (CH1) of a heavy chain. Fab' fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteins from theantibody hinge region. Fab′-SH are Fab′ fragments wherein the cysteineresidue(s) of the constant domains bear a free thiol group. Pepsintreatment yields an F(ab′)2 fragment that has two antigen-combiningsites (two Fab fragments) and a part of the Fc region. According to thepresent invention, the term “Fab fragment” also includes “cross-Fabfragments” or “crossover Fab fragments” as defined below.

The term “cross-Fab fragment” or “xFab fragment” or “crossover Fabfragment” refers to a Fab fragment, wherein either the variable regionsor the constant regions of the heavy and light chain are exchanged. Twodifferent chain compositions of a crossover Fab molecule are possibleand comprised in the bispecific antibodies of the invention: On the onehand, the variable regions of the Fab heavy and light chain areexchanged, i.e. the crossover Fab molecule comprises a peptide chaincomposed of the light chain variable region (VL) and the heavy chainconstant region (CH1), and a peptide chain composed of the heavy chainvariable region (VH) and the light chain constant region (CL). Thiscrossover Fab molecule is also referred to as CrossFab_((VLVH)). On theother hand, when the constant regions of the Fab heavy and light chainare exchanged, the crossover Fab molecule comprises a peptide chaincomposed of the heavy chain variable region (VH) and the light chainconstant region (CL), and a peptide chain composed of the light chainvariable region (VL) and the heavy chain constant region (CH1). Thiscrossover Fab molecule is also referred to as CrossFab_((CLCH1)).

A “single chain Fab fragment” or “scFab” is a polypeptide consisting ofan antibody heavy chain variable domain (VH), an antibody constantdomain 1 (CH1), an antibody light chain variable domain (VL), anantibody light chain constant domain (CL) and a linker, wherein saidantibody domains and said linker have one of the following orders inN-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b)VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL;and wherein said linker is a polypeptide of at least 30 amino acids,preferably between 32 and 50 amino acids. Said single chain Fabfragments are stabilized via the natural disulfide bond between the CLdomain and the CH1 domain. In addition, these single chain Fab moleculesmight be further stabilized by generation of interchain disulfide bondsvia insertion of cysteine residues (e.g. position 44 in the variableheavy chain and position 100 in the variable light chain according toKabat numbering).

A “crossover single chain Fab fragment” or “x-scFab” is a is apolypeptide consisting of an antibody heavy chain variable domain (VH),an antibody constant domain 1 (CH1), an antibody light chain variabledomain (VL), an antibody light chain constant domain (CL) and a linker,wherein said antibody domains and said linker have one of the followingorders in N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH1 andb) VL-CH1-linker-VH-CL; wherein VH and VL form together anantigen-binding site which binds specifically to an antigen and whereinsaid linker is a polypeptide of at least 30 amino acids. In addition,these x-scFab molecules might be further stabilized by generation ofinterchain disulfide bonds via insertion of cysteine residues (e.g.position 44 in the variable heavy chain and position 100 in the variablelight chain according to Kabat numbering).

A “single-chain variable fragment (scFv)” is a fusion protein of thevariable regions of the heavy (V_(H)) and light chains (V_(L)) of anantibody, connected with a short linker peptide of ten to about 25 aminoacids. The linker is usually rich in glycine for flexibility, as well asserine or threonine for solubility, and can either connect theN-terminus of the V_(H) with the C-terminus of the V_(L), or vice versa.This protein retains the specificity of the original antibody, despiteremoval of the constant regions and the introduction of the linker. scFvantibodies are, e.g. described in Houston, J. S., Methods in Enzymol.203 (1991) 46-96). In addition, antibody fragments comprise single chainpolypeptides having the characteristics of a VH domain, namely beingable to assemble together with a VL domain, or of a VL domain, namelybeing able to assemble together with a VH domain to a functional antigenbinding site and thereby providing the antigen binding property of fulllength antibodies.

“Scaffold antigen binding proteins” are known in the art, for example,fibronectin and designed ankyrin repeat proteins (DARPins) have beenused as alternative scaffolds for antigen-binding domains, see, e.g.,Gebauer and Skerra, Engineered protein scaffolds as next-generationantibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumppet al., Darpins: A new generation of protein therapeutics. DrugDiscovery Today 13: 695-701 (2008). In one aspect of the invention, ascaffold antigen binding protein is selected from the group consistingof CTLA-4 (Evibody), Lipocalins (Anticalin), a Protein A-derivedmolecule such as Z-domain of Protein A (Affibody), an A-domain(Avimer/Maxibody), a serum transferrin (trans-body); a designed ankyrinrepeat protein (DARPin), a variable domain of antibody light chain orheavy chain (single-domain antibody, sdAb), a variable domain ofantibody heavy chain (nanobody, aVH), V_(NAR) fragments, a fibronectin(AdNectin), a C-type lectin domain (Tetranectin); a variable domain of anew antigen receptor beta-lactamase (V_(NAR) fragments), a humangamma-crystallin or ubiquitin (Affilin molecules); a kunitz type domainof human protease inhibitors, microbodies such as the proteins from theknottin family, peptide aptamers and fibronectin (adnectin). CTLA-4(Cytotoxic T Lymphocyte-associated Antigen 4) is a CD28-family receptorexpressed on mainly CD4⁺ T-cells. Its extracellular domain has avariable domain-like Ig fold. Loops corresponding to CDRs of antibodiescan be substituted with heterologous sequence to confer differentbinding properties. CTLA-4 molecules engineered to have differentbinding specificities are also known as Evibodies (e.g. U.S. Pat. No.7,166,697B1). Evibodies are around the same size as the isolatedvariable region of an antibody (e.g. a domain antibody). For furtherdetails see Journal of Immunological Methods 248 (1-2), 31-45 (2001).Lipocalins are a family of extracellular proteins which transport smallhydrophobic molecules such as steroids, bilins, retinoids and lipids.They have a rigid beta-sheet secondary structure with a number of loopsat the open end of the conical structure which can be engineered to bindto different target antigens. Anticalins are between 160-180 amino acidsin size, and are derived from lipocalins. For further details seeBiochim Biophys Acta 1482: 337-350 (2000), U.S. Pat. No. 7,250,297B1 andUS20070224633. An affibody is a scaffold derived from Protein A ofStaphylococcus aureus which can be engineered to bind to antigen. Thedomain consists of a three-helical bundle of approximately 58 aminoacids. Libraries have been generated by randomization of surfaceresidues. For further details see Protein Eng. Des. Sel. 2004, 17,455-462 and EP 1641818A1. Avimers are multidomain proteins derived fromthe A-domain scaffold family. The native domains of approximately 35amino acids adopt a defined disulfide bonded structure. Diversity isgenerated by shuffling of the natural variation exhibited by the familyof A-domains. For further details see Nature Biotechnology 23(12),1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6),909-917 (June 2007). A transferrin is a monomeric serum transportglycoprotein. Transferrins can be engineered to bind different targetantigens by insertion of peptide sequences in a permissive surface loop.Examples of engineered transferrin scaffolds include the Trans-body. Forfurther details see J. Biol. Chem 274, 24066-24073 (1999). DesignedAnkyrin Repeat Proteins (DARPins) are derived from Ankyrin which is afamily of proteins that mediate attachment of integral membrane proteinsto the cytoskeleton. A single ankyrin repeat is a 33 residue motifconsisting of two alpha-helices and a beta-turn. They can be engineeredto bind different target antigens by randomizing residues in the firstalpha-helix and a beta-turn of each repeat. Their binding interface canbe increased by increasing the number of modules (a method of affinitymaturation). For further details see J. Mol. Biol. 332, 489-503 (2003),PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007)and US20040132028A1. A single-domain antibody is an antibody fragmentconsisting of a single monomeric variable antibody domain. The firstsingle domains were derived from the variable domain of the antibodyheavy chain from camelids (nanobodies or V_(H)H fragments). Furthermore,the term single-domain antibody includes an autonomous human heavy chainvariable domain (aVH) or V_(NAR) fragments derived from sharks.Fibronectin is a scaffold which can be engineered to bind to antigen.Adnectins consists of a backbone of the natural amino acid sequence ofthe 10th domain of the 15 repeating units of human fibronectin type III(FN3). Three loops at one end of the .beta.-sandwich can be engineeredto enable an Adnectin to specifically recognize a therapeutic target ofinterest. For further details see Protein Eng. Des. Sel. 18, 435-444(2005), US20080139791, WO2005056764 and U.S. Pat. No. 6,818,418B1.Peptide aptamers are combinatorial recognition molecules that consist ofa constant scaffold protein, typically thioredoxin (TrxA) which containsa constrained variable peptide loop inserted at the active site. Forfurther details see Expert Opin. Biol. Ther. 5, 783-797 (2005).Microbodies are derived from naturally occurring microproteins of 25-50amino acids in length which contain 3-4 cysteine bridges—examples ofmicroproteins include KalataBI and conotoxin and knottins. Themicroproteins have a loop which can beengineered to include upto 25amino acids without affecting the overall fold of the microprotein. Forfurther details of engineered knottin domains, see WO2008098796.

An “antigen binding molecule that binds to the same epitope” as areference molecule refers to an antigen binding molecule that blocksbinding of the reference molecule to its antigen in a competition assayby 50% or more, and conversely, the reference molecule blocks binding ofthe antigen binding molecule to its antigen in a competition assay by50% or more.

The term “antigen binding domain” or “antigen-binding site” refers tothe part of an antigen binding molecule that comprises the area whichspecifically binds to and is complementary to part or all of an antigen.Where an antigen is large, an antigen binding molecule may only bind toa particular part of the antigen, which part is termed an epitope. Anantigen binding domain may be provided by, for example, one or morevariable domains (also called variable regions). Preferably, an antigenbinding domain comprises an antibody light chain variable region (VL)and an antibody heavy chain variable region (VH).

As used herein, the term “antigenic determinant” is synonymous with“antigen” and “epitope,” and refers to a site (e.g. a contiguous stretchof amino acids or a conformational configuration made up of differentregions of non-contiguous amino acids) on a polypeptide macromolecule towhich an antigen binding moiety binds, forming an antigen bindingmoiety-antigen complex. Useful antigenic determinants can be found, forexample, on the surfaces of tumor cells, on the surfaces ofvirus-infected cells, on the surfaces of other diseased cells, on thesurface of immune cells, free in blood serum, and/or in theextracellular matrix (ECM). The proteins useful as antigens herein canbe any native form the proteins from any vertebrate source, includingmammals such as primates (e.g. humans) and rodents (e.g. mice and rats),unless otherwise indicated. In a particular embodiment the antigen is ahuman protein. Where reference is made to a specific protein herein, theterm encompasses the “full-length”, unprocessed protein as well as anyform of the protein that results from processing in the cell. The termalso encompasses naturally occurring variants of the protein, e.g.splice variants or allelic variants.

By “specific binding” is meant that the binding is selective for theantigen and can be discriminated from unwanted or non-specificinteractions. The ability of an antigen binding molecule to bind to aspecific antigen can be measured either through an enzyme-linkedimmunosorbent assay (ELISA) or other techniques familiar to one of skillin the art, e.g. Surface Plasmon Resonance (SPR) technique (analyzed ona BlAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)),and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).In one embodiment, the extent of binding of an antigen binding moleculeto an unrelated protein is less than about 10% of the binding of theantigen binding molecule to the antigen as measured, e.g. by SPR. Incertain embodiments, an molecule that binds to the antigen has adissociation constant (Kd) of ≤1μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³M, e.g. from 10⁻⁹ M to 10⁻¹³ M).

“Affinity” or “binding affinity” refers to the strength of the sum totalof non-covalent interactions between a single binding site of a molecule(e.g. an antibody) and its binding partner (e.g. an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g.

antibody and antigen). The affinity of a molecule X for its partner Ycan generally be represented by the dissociation constant (Kd), which isthe ratio of dissociation and association rate constants (koff and kon,respectively). Thus, equivalent affinities may comprise different rateconstants, as long as the ratio of the rate constants remains the same.Affinity can be measured by common methods known in the art, includingthose described herein. A particular method for measuring affinity isSurface Plasmon Resonance (SPR).

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

A “target cell antigen” as used herein refers to an antigenicdeterminant presented on the surface of a target cell, in particular atarget cell in a tumor such as a cancer cell or a cell of the tumorstroma. Thus, the target cell antigen is a tumor-associated antigen. Inparticular, a target cell antigen does not include immune checkpointreceptors on activated T cells, such as CTLA-4, PD-1 or PD-L1. Incertain embodiments, the target cell antigen is an antigen on thesurface of a tumor cell. In one aspect, the tumor target cell antigen isselected from the group consisting of Fibroblast Activation Protein(FAP), Carcinoembryonic Antigen (CEA), Melanoma-associated ChondroitinSulfate Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR),CD19, CD20 and CD33. In particular, the tumor target cell antigen isFibroblast Activation Protein (FAP).

The term “Fibroblast activation protein (FAP)”, also known as Prolylendopeptidase FAP or Seprase (EC 3.4.21), refers to any native FAP fromany vertebrate source, including mammals such as primates (e.g. humans)non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice andrats), unless otherwise indicated. The term encompasses “full-length,”unprocessed FAP as well as any form of FAP that results from processingin the cell. The term also encompasses naturally occurring variants ofFAP, e.g., splice variants or allelic variants. In one embodiment, theantigen binding molecule of the invention is capable of specific bindingto human, mouse and/or cynomolgus FAP. The amino acid sequence of humanFAP is shown in UniProt (www.uniprot.org) accession no. Q12884 (version149, SEQ ID NO:2), or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_004451.2.The extracellular domain (ECD) of human FAP extends from amino acidposition 26 to 760. The amino acid sequence of a His-tagged human FAPECD is shown in SEQ ID NOs 142. The amino acid sequence of mouse FAP isshown in UniProt accession no. P97321 (version 126, SEQ ID NO:143), orNCBI RefSeq NP_032012.1. The extracellular domain (ECD) of mouse FAPextends from amino acid position 26 to 761. SEQ ID NO: 144 shows theamino acid of a His-tagged mouse FAP ECD. SEQ ID NO:145 shows the aminoacid of a His-tagged cynomolgus FAP ECD. Preferably, an anti-FAP bindingmolecule of the invention binds to the extracellular domain of FAP.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding the antigenbinding molecule to antigen. The variable domains of the heavy chain andlight chain (VH and VL, respectively) of a native antibody generallyhave similar structures, with each domain comprising four conservedframework regions (FRs) and three hypervariable regions (HVRs). See,e.g., Kindt et al., Kuby Immunology, 6th ed., W. H. Freeman and Co.,page 91 (2007). A single VH or VL domain may be sufficient to conferantigen-binding specificity.

The term “hypervariable region” or “HVR,” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) Hypervariable regions(HVRs) are also referred to as complementarity determining regions(CDRs), and these terms are used herein interchangeably in reference toportions of the variable region that form the antigen binding regions.This particular region has been described by Kabat et al., U.S. Dept. ofHealth and Human Services, “Sequences of Proteins of ImmunologicalInterest” (1983) and by Chothia et al., J. Mol. Biol. 196:901-917(1987), where the definitions include overlapping or subsets of aminoacid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orvariants thereof is intended to be within the scope of the term asdefined and used herein. The appropriate amino acid residues whichencompass the CDRs as defined by each of the above cited references areset forth below in Table A as a comparison. The exact residue numberswhich encompass a particular CDR will vary depending on the sequence andsize of the CDR. Those skilled in the art can routinely determine whichresidues comprise a particular CDR given the variable region amino acidsequence of the antibody.

TABLE A CDR Definitions¹ CDR Kabat Chothia AbM² V_(H) CDR1 31-35 26-3226-35 V_(H) CDR2 50-65 52-58 50-58 V_(H) CDR3  95-102  95-102  95-102V_(L) CDR1 24-34 26-32 24-34 V_(L) CDR2 50-56 50-52 50-56 V_(L) CDR389-97 91-96 89-97 ¹Numbering of all CDR definitions in Table A isaccording to the numbering conventions set forth by Kabat et al. (seebelow). ²“AbM” with a lowercase “b” as used in Table A refers to theCDRs as defined by Oxford Molecular's “AbM” antibody modeling software.

Kabat et al. also defined a numbering system for variable regionsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable region sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody variable region areaccording to the Kabat numbering system.

With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. CDRs also comprise“specificity determining residues,” or “SDRs,” which are residues thatcontact antigen. SDRs are contained within regions of the CDRs calledabbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2,a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633(2008).) Unless otherwise indicated, HVR residues and other residues inthe variable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g. IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ respectively.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization. Other forms of “humanized antibodies” encompassed by thepresent invention are those in which the constant region has beenadditionally modified or changed from that of the original antibody togenerate the properties according to the invention, especially in regardto C1q binding and/or Fc receptor (FcR) binding.

A “human” antibody is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an antibody heavy chain that contains at least aportion of the constant region. The term includes native sequence Fcregions and variant Fc regions. An IgG Fc region comprises an IgG CH2and an IgG CH3 domain. The “CH2 domain” of a human IgG Fc region usuallyextends from an amino acid residue at about position 231 to an aminoacid residue at about position 340. In one embodiment, a carbohydratechain is attached to the CH2 domain. The CH2 domain herein may be anative sequence CH2 domain or variant CH2 domain. The “CH3 domain”comprises the stretch of residues C-terminal to a CH2 domain in an Fcregion (i.e. from an amino acid residue at about position 341 to anamino acid residue at about position 447 of an IgG). The CH3 regionherein may be a native sequence CH3 domain or a variant CH3 domain (e.g.a CH3 domain with an introduced “protuberance” (“knob”) in one chainthereof and a corresponding introduced “cavity” (“hole”) in the otherchain thereof; see U.S. Pat. No. 5,821,333, expressly incorporatedherein by reference). Such variant CH3 domains may be used to promoteheterodimerization of two non-identical antibody heavy chains as hereindescribed. In one embodiment, a human IgG heavy chain Fc region extendsfrom Cys226, or from Pro230, to the carboxyl-terminus of the heavychain. However, the C-terminal lysine (Lys447) of the Fc region may ormay not be present. Unless otherwise specified herein, numbering ofamino acid residues in the Fc region or constant region is according tothe EU numbering system, also called the EU index, as described in Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., 1991.

The “knob-into-hole” technology is described e.g. in U.S. Pat. No.5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621(1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, themethod involves introducing a protuberance (“knob”) at the interface ofa first polypeptide and a corresponding cavity (“hole”) in the interfaceof a second polypeptide, such that the protuberance can be positioned inthe cavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). The protuberance and cavitycan be made by altering the nucleic acid encoding the polypeptides, e.g.by site-specific mutagenesis, or by peptide synthesis. In a specificembodiment a knob modification comprises the amino acid substitutionT366W in one of the two subunits of the Fc domain, and the holemodification comprises the amino acid substitutions T366S, L368A andY407V in the other one of the two subunits of the Fc domain. In afurther specific embodiment, the subunit of the Fc domain comprising theknob modification additionally comprises the amino acid substitutionS354C, and the subunit of the Fc domain comprising the hole modificationadditionally comprises the amino acid substitution Y349C. Introductionof these two cysteine residues results in the formation of a disulfidebridge between the two subunits of the Fc region, thus furtherstabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

A “region equivalent to the Fc region of an immunoglobulin” is intendedto include naturally occurring allelic variants of the Fc region of animmunoglobulin as well as variants having alterations which producesubstitutions, additions, or deletions but which do not decreasesubstantially the ability of the immunoglobulin to mediate effectorfunctions (such as antibody-dependent cellular cytotoxicity). Forexample, one or more amino acids can be deleted from the N-terminus orC-terminus of the Fc region of an immunoglobulin without substantialloss of biological function. Such variants can be selected according togeneral rules known in the art so as to have minimal effect on activity(see, e.g., Bowie, J. U. et al., Science 247:1306-10 (1990)).

The term “effector function” refers to those biological activitiesattributable to the Fc region of an antibody, which vary with theantibody isotype. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC), Fc receptorbinding, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokine secretion,immune complex-mediated antigen uptake by antigen presenting cells, downregulation of cell surface receptors (e.g. B cell receptor), and B cellactivation.

Fc receptor binding dependent effector functions can be mediated by theinteraction of the Fc-region of an antibody with Fc receptors (FcRs),which are specialized cell surface receptors on hematopoietic cells. Fcreceptors belong to the immunoglobulin superfamily, and have been shownto mediate both the removal of antibody-coated pathogens by phagocytosisof immune complexes, and the lysis of erythrocytes and various othercellular targets (e.g. tumor cells) coated with the correspondingantibody, via antibody dependent cell mediated cytotoxicity (ADCC) (seee.g. Van de Winkel, J. G. and Anderson, C. L., J. Leukoc. Biol. 49(1991) 511-524). FcRs are defined by their specificity forimmunoglobulin isotypes: Fc receptors for IgG antibodies are referred toas FcγR. Fc receptor binding is described e.g. in Ravetch, J. V. andKinet, J. P., Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P. J., etal., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J. Lab. Clin.Med. 126 (1995) 330-341; and Gessner, J. E., et al., Ann. Hematol. 76(1998) 231-248.

Cross-linking of receptors for the Fc-region of IgG antibodies (FcγR)triggers a wide variety of effector functions including phagocytosis,antibody-dependent cellular cytotoxicity, and release of inflammatorymediators, as well as immune complex clearance and regulation ofantibody production. In humans, three classes of FcγR have beencharacterized, which are:

FcγRI (CD64) binds monomeric IgG with high affinity and is expressed onmacrophages, monocytes, neutrophils and eosinophils. Modification in theFc-region IgG at least at one of the amino acid residues E233-G236,P238, D265, N297, A327 and P329 (numbering according to EU index ofKabat) reduce binding to FcγRI. IgG2 residues at positions 233-236,substituted into IgG1 and IgG4, reduced binding to FcγRI by 10³-fold andeliminated the human monocyte response to antibody-sensitized red bloodcells (Armour, K. L., et al., Eur. J. Immunol. 29 (1999) 2613-2624).

-FcγRII (CD32) binds complexed IgG with medium to low affinity and iswidely expressed. This receptor can be divided into two sub-types,FcγRIIA and FcγRIIB FcγRIIA is found on many cells involved in killing(e.g. macrophages, monocytes, neutrophils) and seems able to activatethe killing process. FcγRIIB seems to play a role in inhibitoryprocesses and is found on B cells, macrophages and on mast cells andeosinophils. On B-cells it seems to function to suppress furtherimmunoglobulin production and isotype switching to, for example, the IgEclass. On macrophages, FcγRIIB acts to inhibit phagocytosis as mediatedthrough FcγRIIA. On eosinophils and mast cells the B-form may help tosuppress activation of these cells through IgE binding to its separatereceptor. Reduced binding for FcγRIIA is found e.g. for antibodiescomprising an IgG Fc-region with mutations at least at one of the aminoacid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327,R292, and K414 (numbering according to EU index of Kabat).

FcγRIII (CD16) binds IgG with medium to low affinity and exists as twotypes. FcγRIIIA is found on NK cells, macrophages, eosinophils and somemonocytes and T cells and mediates ADCC. FcγRIIIB is highly expressed onneutrophils. Reduced binding to FcγRIIIA is found e.g. for antibodiescomprising an IgG Fc-region with mutation at least at one of the aminoacid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327,5239, E269, E293, Y296, V303, A327, K338 and D376 (numbering accordingto EU index of Kabat).

Mapping of the binding sites on human IgG1 for Fc receptors, the abovementioned mutation sites and methods for measuring binding to FcγRI andFcγRIIA are described in Shields, R. L., et al. J. Biol. Chem. 276(2001) 6591-6604.

The term “ADCC” or “antibody-dependent cellular cytotoxicity” is afunction mediated by Fc receptor binding and refers to lysis of targetcells by an antibody as reported herein in the presence of effectorcells. The capacity of the antibody to induce the initial stepsmediating ADCC is investigated by measuring their binding to Feyreceptors expressing cells, such as cells, recombinantly expressingFcγRI and/or FcγRIIA or NK cells (expressing essentially FcγRIIIA) Inparticular, binding to FcγR on NK cells is measured.

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc region of an antibody elicits signaling events that stimulatethe receptor-bearing cell to perform effector functions. Activating Fcreceptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), andFcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (seeUniProt accession no. P08637, version 141).

The term “CD40”, as used herein, refers to any native CD40 from anyvertebrate source, including mammals such as primates (e.g. humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed CD40 as well as any form of CD40that results from processing in the cell. The term also encompassesnaturally occurring variants of CD40, e.g., splice variants or allelicvariants. The amino acid sequence of an exemplary human CD40 is shown inSEQ ID NO:1 (Uniprot P25942, version 200) and the amino acid sequence ofan exemplary mouse CD40 is shown in SEQ ID NO: 146 (Uniprot P27512,version 160). The CD40 antigen is a 50 kDa cell surface glycoproteinwhich belongs to the Tumor Necrosis Factor Receptor (TNF-R) family.(Stamenkovic et al. (1989), EMBO J. 8: 1403-10). CD40 is expressed inmany normal and tumor cell types, including B lymphocytes, dendriticcells, monocytes, macrophages, thymus epithelium, endothelial cells,fibroblasts, and smooth muscle cells. CD40 is expressed in allB-lymphomas and in 70% of all solid tumors and is up-regulated inantigen presenting cells (APCs) by maturation signals, such as IFN-gammaand GM-CSF. CD40 activation also induces differentiation of monocytesinto functional dendritic cells (DCs) and enhances cytolytic activity ofNK cells through APC-CD40 induced cytokines. Thus CD40 plays anessential role in the initiation and enhancement of immune responses byinducing maturation of APCs, secretion of helper cytokines, upregulationof costimulatory molecules, and enhancement of effector functions.

The term “CD40 agonist” as used herein includes any moiety that agonizesthe CD40/CD40L interaction. CD40 as used in this context referspreferably to human CD40, thus the CD40 agonist is preferably an agonistof human CD40. Typically, the moiety will be an agonistic CD40 antibodyor antibody fragment.

The terms “anti-CD40 antibody”, “anti-CD40”, “CD40 antibody and “anantibody that specifically binds to CD40” refer to an antibody that iscapable of binding CD40 with sufficient affinity such that the antibodyis useful as a diagnostic and/or therapeutic agent in targeting CD40. Inone aspect, the extent of binding of an anti-CD40 antibody to anunrelated, non-CD40 protein is less than about 10% of the binding of theantibody to CD40 as measured, e.g., by a radioimmunoassay (RIA) or flowcytometry (FACS). In certain embodiments, an antibody that binds to CD40has a dissociation constant (K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM,≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁶M or less, e.g. from 10⁻⁶⁸M to10¹³M, e.g., from 10⁻⁸M to 10⁻¹⁰ M).

The term “peptide linker” refers to a peptide comprising one or moreamino acids, typically about 2 to 20 amino acids. Peptide linkers areknown in the art or are described herein. Suitable, non-immunogeniclinker peptides are, for example, (G₄S)_(n), (SG₄)_(n) or G₄(SG₄)_(n)peptide linkers, wherein “n” is generally a number between 1 and 10,typically between 2 and 4, in particular 2, i.e. the peptides selectedfrom the group consisting of GGGGS (SEQ ID NO: 147) GGGGSGGGGS (SEQ IDNO:148), SGGGGSGGGG (SEQ ID NO:149) and GGGGSGGGGSGGGG (SEQ ID NO:150),but also include the sequences GSPGSSSSGS (SEQ ID NO:151), (G4S)3 (SEQID NO:152), (G4S)₄ (SEQ ID NO:153), GSGSGSGS (SEQ ID NO:154), GSGSGNGS(SEQ ID NO:155), GGSGSGSG (SEQ ID NO:156), GGSGSG (SEQ ID NO:157), GGSG(SEQ ID NO:158), GGSGNGSG (SEQ ID NO:159), GGNGSGSG (SEQ ID NO:160) andGGNGSG (SEQ ID NO:161). Peptide linkers of particular interest are (G4S)(SEQ ID NO:147), (G₄S)₂ or GGGGSGGGGS (SEQ ID NO:148), (G4S)₃ (SEQ IDNO:152) and (G4S)₄ (SEQ ID NO:153).

The term “amino acid” as used within this application denotes the groupof naturally occurring carboxy a-amino acids comprising alanine (threeletter code: ala, one letter code: A), arginine (arg, R), asparagine(asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q),glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine(ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M),phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine(thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

By “fused” or “connected” is meant that the components (e.g. a heavychain of an antibody and a Fab fragment) are linked by peptide bonds,either directly or via one or more peptide linkers.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide (protein) sequence is defined as the percentage of aminoacid residues in a candidate sequence that are identical with the aminoacid residues in the reference polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN. SAWIor Megalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for aligning sequences, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program ALIGN-2. The ALIGN-2 sequence comparison computerprogram was authored by Genentech, Inc., and the source code has beenfiled with user documentation in the U.S. Copyright Office, WashingtonD.C., 20559, where it is registered under U.S. Copyright RegistrationNo. TXU510087. The ALIGN-2 program is publicly available from Genentech,Inc., South San Francisco, Calif., or may be compiled from the sourcecode. The ALIGN-2 program should be compiled for use on a UNIX operatingsystem, including digital UNIX V4.0D. All sequence comparison parametersare set by the ALIGN-2 program and do not vary. In situations whereALIGN-2 is employed for amino acid sequence comparisons, the % aminoacid sequence identity of a given amino acid sequence A to, with, oragainst a given amino acid sequence B (which can alternatively bephrased as a given amino acid sequence A that has or comprises a certain% amino acid sequence identity to, with, or against a given amino acidsequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

In certain embodiments, amino acid sequence variants of the bispecificantigen binding molecules provided herein are contemplated. For example,it may be desirable to improve the binding affinity and/or otherbiological properties of the TNF ligand trimer-containing antigenbinding molecules. Amino acid sequence variants of the TNF ligandtrimer-containing antigen binding molecules may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the molecules, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.Sites of interest for substitutional mutagenesis include the HVRs andFramework (FRs). Conservative substitutions are provided in Table Bunder the heading “Preferred Substitutions” and further described belowin reference to amino acid side chain classes (1) to (6). Amino acidsubstitutions may be introduced into the molecule of interest and theproducts screened for a desired activity, e.g., retained/improvedantigen binding, decreased immunogenicity, or improved ADCC or CDC.

TABLE B Original Preferred Residue Exemplary Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

The term “amino acid sequence variants” includes substantial variantswherein there are amino acid substitutions in one or more hypervariableregion residues of a parent antigen binding molecule (e.g. a humanizedor human antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antigen binding molecule and/or will havesubstantially retained certain biological properties of the parentantigen binding molecule. An exemplary substitutional variant is anaffinity matured antibody, which may be conveniently generated, e.g.,using phage display-based affinity maturation techniques such as thosedescribed herein. Briefly, one or more HVR residues are mutated and thevariant antigen binding molecules displayed on phage and screened for aparticular biological activity (e.g. binding affinity). In certainembodiments, substitutions, insertions, or deletions may occur withinone or more HVRs so long as such alterations do not substantially reducethe ability of the antigen binding molecule to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. A useful method for identification of residues orregions of an antibody that may be targeted for mutagenesis is called“alanine scanning mutagenesis” as described by Cunningham and Wells(1989) Science, 244:1081-1085. In this method, a residue or group oftarget residues (e.g., charged residues such as Arg, Asp, His, Lys, andGlu) are identified and replaced by a neutral or negatively chargedamino acid (e.g., alanine or polyalanine) to determine whether theinteraction of the antibody with antigen is affected. Furthersubstitutions may be introduced at the amino acid locationsdemonstrating functional sensitivity to the initial substitutions.Alternatively, or additionally, a crystal structure of anantigen-antigen binding molecule complex to identify contact pointsbetween the antibody and antigen. Such contact residues and neighboringresidues may be targeted or eliminated as candidates for substitution.Variants may be screened to determine whether they contain the desiredproperties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includebispecific antigen binding molecules of the invention with an N-terminalmethionyl residue. Other insertional variants of the molecule includethe fusion to the N- or C-terminus to a polypeptide which increases theserum half-life of the bispecific antigen binding molecules.

In certain embodiments, the bispecific antigen binding moleculesprovided herein are altered to increase or decrease the extent to whichthe antibody is glycosylated. Glycosylation variants of the moleculesmay be conveniently obtained by altering the amino acid sequence suchthat one or more glycosylation sites is created or removed. Where theTNF ligand trimer-containing antigen binding molecule comprises an Fcregion, the carbohydrate attached thereto may be altered. Nativeantibodies produced by mammalian cells typically comprise a branched,biantennary oligosaccharide that is generally attached by an N-linkageto Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al.TIBTECH 15:26-32 (1997). The oligosaccharide may include variouscarbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose,and sialic acid, as well as a fucose attached to a GlcNAc in the “stem”of the biantennary oligosaccharide structure. In some embodiments,modifications of the oligosaccharide in TNF family ligandtrimer-containing antigen binding molecule may be made in order tocreate variants with certain improved properties. In one aspect,variants of bispecific antigen binding molecules or antibodies of theinvention are provided having a carbohydrate structure that lacks fucoseattached (directly or indirectly) to an Fc region. Such fucosylationvariants may have improved ADCC function, see e.g. US Patent PublicationNos. US 2003/0157108 (Presta, L.) or US 2004/0093621 (Kyowa Hakko KogyoCo., Ltd). In another aspect, variants of the bispecific antigen bindingmolecules or antibodies of the invention are provided with bisectedoligosaccharides, e.g., in which a biantennary oligosaccharide attachedto the Fc region is bisected by GlcNAc. Such variants may have reducedfucosylation and/or improved ADCC function., see for example WO2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana etal.); and US 2005/0123546 (Umana et al.). Variants with at least onegalactose residue in the oligosaccharide attached to the Fc region arealso provided. Such antibody variants may have improved CDC function andare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

In certain aspects, it may be desirable to create cysteine engineeredvariants of the bispecific antigen binding molecules of the invention,e.g., “thioMAbs,” in which one or more residues of the molecule aresubstituted with cysteine residues. In particular aspects, thesubstituted residues occur at accessible sites of the molecule. Bysubstituting those residues with cysteine, reactive thiol groups arethereby positioned at accessible sites of the antibody and may be usedto conjugate the antibody to other moieties, such as drug moieties orlinker-drug moieties, to create an immunoconjugate. In certain aspects,any one or more of the following residues may be substituted withcysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering)of the heavy chain; and S400 (EU numbering) of the heavy chain Fcregion. Cysteine engineered antigen binding molecules may be generatedas described, e.g., in U.S. Pat. No. 7,521,541.

The term “polynucleotide” refers to an isolated nucleic acid molecule orconstruct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmidDNA (pDNA). A polynucleotide may comprise a conventional phosphodiesterbond or a non-conventional bond (e.g. an amide bond, such as found inpeptide nucleic acids (PNA). The term “nucleic acid molecule” refers toany one or more nucleic acid segments, e.g. DNA or RNA fragments,present in a polynucleotide.

By “isolated” nucleic acid molecule or polynucleotide is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encoding apolypeptide contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. An isolated polynucleotide includes apolynucleotide molecule contained in cells that ordinarily contain thepolynucleotide molecule, but the polynucleotide molecule is presentextrachromosomally or at a chromosomal location that is different fromits natural chromosomal location. Isolated RNA molecules include in vivoor in vitro RNA transcripts of the present invention, as well aspositive and negative strand forms, and double-stranded forms. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. In addition, apolynucleotide or a nucleic acid may be or may include a regulatoryelement such as a promoter, ribosome binding site, or a transcriptionterminator.

By a nucleic acid or polynucleotide having a nucleotide sequence atleast, for example, 95% “identical” to a reference nucleotide sequenceof the present invention, it is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence. In otherwords, to obtain a polynucleotide having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at the5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. As a practical matter,whether any particular polynucleotide sequence is at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of thepresent invention can be determined conventionally using known computerprograms, such as the ones discussed above for polypeptides (e.g.ALIGN-2).

The term “expression cassette” refers to a polynucleotide generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in atarget cell. The recombinant expression cassette can be incorporatedinto a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, ornucleic acid fragment. Typically, the recombinant expression cassetteportion of an expression vector includes, among other sequences, anucleic acid sequence to be transcribed and a promoter. In certainembodiments, the expression cassette of the invention comprisespolynucleotide sequences that encode bispecific antigen bindingmolecules of the invention or fragments thereof.

The term “vector” or “expression vector” is synonymous with “expressionconstruct” and refers to a DNA molecule that is used to introduce anddirect the expression of a specific gene to which it is operablyassociated in a target cell. The term includes the vector as aself-replicating nucleic acid structure as well as the vectorincorporated into the genome of a host cell into which it has beenintroduced. The expression vector of the present invention comprises anexpression cassette. Expression vectors allow transcription of largeamounts of stable mRNA. Once the expression vector is inside the targetcell, the ribonucleic acid molecule or protein that is encoded by thegene is produced by the cellular transcription and/or translationmachinery. In one embodiment, the expression vector of the inventioncomprises an expression cassette that comprises polynucleotide sequencesthat encode bispecific antigen binding molecules of the invention orfragments thereof.

The terms “host cell”, “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.A host cell is any type of cellular system that can be used to generatethe bispecific antigen binding molecules of the present invention. Hostcells include cultured cells, e.g. mammalian cultured cells, such as CHOcells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mousemyeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells,insect cells, and plant cells, to name only a few, but also cellscomprised within a transgenic animal, transgenic plant or cultured plantor animal tissue.

An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). Particularly, the individualor subject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable excipient” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable excipient includes,but is not limited to, a buffer, a stabilizer, or a preservative.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, the moleculesof the invention are used to delay development of a disease or to slowthe progression of a disease.

The term “cancer” as used herein refers to proliferative diseases, suchas lymphomas, lymphocytic leukemias, lung cancer, non-small cell lung(NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, gastric cancer, colon cancer,breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, prostate cancer, cancer of the bladder,cancer of the kidney or ureter, renal cell carcinoma, carcinoma of therenal pelvis, mesothelioma, hepatocellular cancer, biliary cancer,neoplasms of the central nervous system (CNS), spinal axis tumors, brainstem glioma, glioblastoma multiforme, astrocytomas, schwanomas,ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas,pituitary adenoma and Ewings sarcoma, including refractory versions ofany of the above cancers, or a combination of one or more of the abovecancers.

The term “chemotherapeutic agent” as used herein refers to a chemicalcompound useful in the treatment of cancer. In one aspect, thechemotherapeutic agent is an antimetabolite. In one aspect, theantimetabolite is selected from the group consisting of Aminopterin,Methotrexate, Pemetrexed, Raltitrexed, Cladribine, Clofarabine,Fludarabine, Mercaptopurine, Pentostatin, Thioguanine, Capecitabine,Cytarabine, Fluorouracil, Floxuridine, and Gemcitabine. In oneparticular aspect, the antimetabolite is capecitabine or gemcitabine. Inanother aspect, the antimetabolite is fluorouracil. In one aspect, thechemotherapeutic agent is an agent that affects microtubule formation.In one aspect, the agent that affects microtubule formation is selectedfrom the group consisting of: paclitaxel, docetaxel, vincristine,vinblastine, vindesine, vinorelbin, taxotere, etoposide, and teniposide.In another aspect, the chemotherapeutic agent is an alkylating agentsuch as cyclophosphamide. In one aspect, the chemotherapeutic agent is acytotoxic antibiotic such as a topoisomerase II inhibitor. In oneaspect, the topoisomerase II inhibitor is doxorubicin.

Bispecific Antibodies of the Invention

The invention provides novel bispecific antigen binding molecules withparticularly advantageous properties such as producibility, stability,binding affinity, biological activity, targeting efficiency, reducedtoxicity, an extended dosage range that can be given to a patient andthereby a possibly enhanced efficacy.

Exemplary Bispecific Antigen Binding Molecules

In one aspect, the invention provides bispecific antigen bindingmolecules, comprising

-   (a) at least one antigen binding domain capable of specific binding    to CD40, and-   (b) at least one antigen binding domain capable of specific binding    to a target cell antigen, and-   (c) a Fc region composed of a first and a second subunit capable of    stable association.

In a particular aspect, these bispecific antigen binding molecules arecharacterized by targeted agonistic binding to CD40. In particular, thebispecific antigen binding molecule is a CD40 agonist that is targetedagainst a tumor associated target cell antigen. In another particularaspect, the bispecific antigen binding molecules of the inventioncomprise a Fc region composed of a first and a second subunit capable ofstable association which comprises mutations that reduce effectorfunction. The use of a Fc region comprising mutations that reduce orabolish effector function will prevent unspecific agonism bycrosslinking via Fc receptors and will prevent ADCC of CD40⁺ cells.

The bispecific antigen binding molecules as described herein possess theadvantage over conventional antibodies capable of specific binding toCD40 in that they selectively induce immune response at the targetcells, which are typically cancer cells or tumor stroma. In one aspect,the tumor-associated target cell antigen is selected from the groupconsisting of Fibroblast Activation Protein (FAP), Melanoma-associatedChondroitin Sulfate Proteoglycan (MCSP), Epidermal Growth FactorReceptor (EGFR), Carcinoembryonic Antigen (CEA), CD19, CD20 and CD33.

In a particular aspect, the tumor-associated target cell antigen is FAP.

These bispecific antigen binding molecules are characterized byFAP-targeted agonistic binding to CD40. In the presence ofFAP-expressing cells the bispecific antigen binding molecules are ableto activate antigen presenting cells (APCs, Example 2.1), to activatehuman B cells (Examples 2.1.1 and 2.1.3), human Daudi cells (Example2.1.2) and human monocyte-derived dendritic cells (moDCs, Example 2.1.4)

In one aspect, provided is a bispecific antigen binding molecule,wherein the antigen binding domain capable of specific binding to CD40comprises a heavy chain variable region (V_(H)CD40) comprising (i)CDR-H1 comprising the amino acid sequence of SEQ ID NO:19, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:20, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:21, and a light chainvariable region (V_(L)CD40) comprising (iv) CDR-L1 comprising the aminoacid sequence of SEQ ID NO:22, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO:23, and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO:24.

In another aspect, provided is a bispecific antigen binding molecule,wherein the antigen binding domain capable of specific binding to CD40comprises a heavy chain variable region (V_(H)CD40) comprising (i)CDR-H1 comprising the amino acid sequence of SEQ ID NO:27, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:28, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:29, and a light chainvariable region (V_(L)CD40) comprising (iv) CDR-L1 comprising the aminoacid sequence of SEQ ID NO:30, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO:31, and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO:32.

In one aspect, the bispecific antigen binding molecule comprises anantigen binding domain capable of specific binding to CD40 and comprisesa comprises a heavy chain variable region (V_(H)CD40) comprising theamino acid sequence of SEQ ID NO:25 and a light chain variable region(V_(L)CD40) comprising the amino acid sequence of SEQ ID NO:26.

In another aspect, the bispecific antigen binding molecule comprises anantigen binding domain capable of specific binding to CD40 and comprisesa comprises a heavy chain variable region (V_(H)CD40) comprising theamino acid sequence of SEQ ID NO:33 and a light chain variable region(V_(L)CD40) comprising the amino acid sequence of SEQ ID NO:34.

In another aspect, provided is a bispecific antigen binding molecule,wherein the antigen binding domain capable of specific binding to CD40comprises

(i) a heavy chain variable region (V_(H)CD40) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55, and

(ii) a light chain variable region (V_(L)CD40) comprising the amino acidsequence selected from the group consisting of SEQ ID NO:56, SEQ IDNO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ IDNO:62, SEQ ID NO:63 and SEQ ID NO:64.

In one aspect, provided is a bispecific antigen binding molecule,wherein the antigen binding domain capable of specific binding to CD40comprises a heavy chain variable region (V_(H)CD40) comprising the aminoacid sequence of SEQ ID NO:47 and a light chain variable region(V_(L)CD40) comprising the amino acid sequence of SEQ ID NO:57.

In a further aspect, provided is a bispecific antigen binding molecule,wherein the antigen binding domain capable of specific binding to CD40comprises

(i) a heavy chain variable region (V_(H)CD40) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:171, SEQ IDNO:172, SEQ ID NO:173 and SEQ ID NO:174, and

(ii) a light chain variable region (V_(L)CD40) comprising the amino acidsequence selected from the group consisting of SEQ ID NO:175, SEQ IDNO:176, SEQ ID NO:177, and SEQ ID NO:178.

In one aspect, provided is a bispecific antigen binding molecule,wherein the antigen binding domain capable of specific binding to CD40comprises

(a) a VH comprising the amino acid sequence of SEQ ID NO:171 and a VLcomprising the amino acid sequence of SEQ ID NO:175, or

(b) a VH comprising the amino acid sequence of SEQ ID NO:173 and a VLcomprising the amino acid sequence of SEQ ID NO:177, or

(c) a VH comprising the amino acid sequence of SEQ ID NO:174 and a VLcomprising the amino acid sequence of SEQ ID NO:178, or

(d) a VH comprising the amino acid sequence of SEQ ID NO:171 and a VLcomprising the amino acid sequence of SEQ ID NO:177, or

(e) a VH comprising the amino acid sequence of SEQ ID NO:171 and a VLcomprising the amino acid sequence of SEQ ID NO:178, or

(f) a VH comprising the amino acid sequence of SEQ ID NO:173 and a VLcomprising the amino acid sequence of SEQ ID NO:175, or

(g) a VH comprising the amino acid sequence of SEQ ID NO:173 and a VLcomprising the amino acid sequence of SEQ ID NO:178, or

(h) a VH comprising the amino acid sequence of SEQ ID NO:174 and a VLcomprising the amino acid sequence of SEQ ID NO:175, or

(i) a VH comprising the amino acid sequence of SEQ ID NO:174 and a VLcomprising the amino acid sequence of SEQ ID NO:177, or

(j) a VH comprising the amino acid sequence of SEQ ID NO:171 and a VLcomprising the amino acid sequence of SEQ ID NO:176, or

(k) a VH comprising the amino acid sequence of SEQ ID NO:172 and a VLcomprising the amino acid sequence of SEQ ID NO:175, or

(l) a VH comprising the amino acid sequence of SEQ ID NO:172 and a VLcomprising the amino acid sequence of SEQ ID NO:176, or

(m) a VH comprising the amino acid sequence of SEQ ID NO:172 and a VLcomprising the amino acid sequence of SEQ ID NO:177, or

(n) a VH comprising the amino acid sequence of SEQ ID NO:172 and a VLcomprising the amino acid sequence of SEQ ID NO:178, or

(o) a VH comprising the amino acid sequence of SEQ ID NO:173 and a VLcomprising the amino acid sequence of SEQ ID NO:176, or

(p) a VH comprising the amino acid sequence of SEQ ID NO:174 and a VLcomprising the amino acid sequence of SEQ ID NO:176.

In a particular aspect, provided is a bispecific antigen bindingmolecule, wherein the antigen binding domain capable of specific bindingto CD40 comprises a VH comprising the amino acid sequence of SEQ IDNO:171 and a VL comprising the amino acid sequence of SEQ ID NO:175.

In yet another aspect, provided is a bispecific antigen bindingmolecule, wherein the antigen binding domain capable of specific bindingto CD40 comprises

(i) a heavy chain variable region (V_(H)CD40) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:179, SEQ IDNO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183 and SEQ ID NO:184,and

(ii) a light chain variable region (V_(L)CD40) comprising the amino acidsequence selected from the group consisting of SEQ ID NO:185, SEQ IDNO:186, SEQ ID NO:187, and SEQ ID NO:188.

In one aspect, provided is a bispecific antigen binding molecule,wherein the antigen binding domain capable of specific binding to CD40comprises

(a) a VH comprising the amino acid sequence of SEQ ID NO:179 and a VLcomprising the amino acid sequence of SEQ ID NO:185, or

(b) a VH comprising the amino acid sequence of SEQ ID NO:180 and a VLcomprising the amino acid sequence of SEQ ID NO:185, or

(c) a VH comprising the amino acid sequence of SEQ ID NO:181 and a VLcomprising the amino acid sequence of SEQ ID NO:185, or

(d) a VH comprising the amino acid sequence of SEQ ID NO:182 and a VLcomprising the amino acid sequence of SEQ ID NO:185, or

(e) a VH comprising the amino acid sequence of SEQ ID NO:179 and a VLcomprising the amino acid sequence of SEQ ID NO:186, or

(f) a VH comprising the amino acid sequence of SEQ ID NO:180 and a VLcomprising the amino acid sequence of SEQ ID NO:186, or

(g) a VH comprising the amino acid sequence of SEQ ID NO:181 and a VLcomprising the amino acid sequence of SEQ ID NO:186, or

(h) a VH comprising the amino acid sequence of SEQ ID NO:182 and a VLcomprising the amino acid sequence of SEQ ID NO:186, or

(i) a VH comprising the amino acid sequence of SEQ ID NO:183 and a VLcomprising the amino acid sequence of SEQ ID NO:187, or

(j) a VH comprising the amino acid sequence of SEQ ID NO:183 and a VLcomprising the amino acid sequence of SEQ ID NO:188, or

(k) a VH comprising the amino acid sequence of SEQ ID NO:184 and a VLcomprising the amino acid sequence of SEQ ID NO:187, or

(l) a VH comprising the amino acid sequence of SEQ ID NO:184 and a VLcomprising the amino acid sequence of SEQ ID NO:188.

In a particular aspect, provided is a bispecific antigen bindingmolecule, wherein the antigen binding domain capable of specific bindingto CD40 comprises a VH comprising the amino acid sequence of SEQ IDNO:179 and a VL comprising the amino acid sequence of SEQ ID NO:185 orwherein the antigen binding domain capable of specific binding to CD40comprises a VH comprising the amino acid sequence of SEQ ID NO:182 and aVL comprising the amino acid sequence of SEQ ID NO:185.

Bispecific Antigen Binding Molecules wherein the Target Cell Antigen isFAP

In a particular aspect, the target cell antigen is Fibroblast ActivationProtein (FAP). FAP binding moieties have been described in WO 2012/02006which is included by reference in its entirety. FAP binding moieties ofparticular interest are described below.

In one aspect, the invention provides a bispecific antigen bindingmolecule, wherein the antigen binding domain capable of specific bindingto FAP binds to a polypeptide comprising, or consisting of, the aminoacid sequence of SEQ ID NO:2.

In another aspect, the invention provides a bispecific antigen bindingmolecule, wherein the antigen binding domain capable of specific bindingto Fibroblast Activation Protein (FAP) comprises

-   (a) a heavy chain variable region (V_(H)FAP) comprising (i) CDR-H1    comprising the amino acid sequence of SEQ ID NO:3, (ii) CDR-H2    comprising the amino acid sequence of SEQ ID NO:4, and (iii) CDR-H3    comprising the amino acid sequence of SEQ ID NO:5, and a light chain    variable region (V_(L)FAP) comprising (iv) CDR-L1 comprising the    amino acid sequence of SEQ ID NO:6, (v) CDR-L2 comprising the amino    acid sequence of SEQ ID NO:7, and (vi) CDR-L3 comprising the amino    acid sequence of SEQ ID NO:8, or-   (b) a heavy chain variable region (V_(H)FAP) comprising (i) CDR-H1    comprising the amino acid sequence of SEQ ID NO:11, (ii) CDR-H2    comprising the amino acid sequence of SEQ ID NO:12, and (iii) CDR-H3    comprising the amino acid sequence of SEQ ID NO:13, and a a light    chain variable region (V_(L)FAP) comprising (iv) CDR-L1 comprising    the amino acid sequence of SEQ ID NO:14, (v) CDR-L2 comprising the    amino acid sequence of SEQ ID NO:15, and (vi) CDR-L3 comprising the    amino acid sequence of SEQ ID NO:16.

In particular, provided is a bispecific antigen binding molecule,wherein the heavy chain variable region (V_(H)FAP) comprising (i) CDR-H1comprising the amino acid sequence of SEQ ID NO:3, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:4, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:5, and the light chainvariable region (V_(L)FAP) comprising (iv) CDR-L1 comprising the aminoacid sequence of SEQ ID NO:6, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO:7, and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO:8. In another aspect, the antigen binding domaincapable of specific binding to FAP comprises a heavy chain variableregion (V_(H)FAP) comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:11, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:12, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ

ID NO:13, and a a light chain variable region (V_(L)FAP) comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO:14, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:15, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:16.

In a further aspect, provided is a bispecific antigen binding molecule,wherein the antigen binding domain capable of specific binding to FAPcomprises (a) a heavy chain variable region (V_(H)FAP) comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO:9, and a lightchain variable region (V_(L)FAP) comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO:10, or (b) a heavy chain variable region(V_(H)FAP) comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO:17, and a light chain variable region (V_(L)FAP) comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO:18.

In one aspect, the antigen binding domain capable of specific binding toFAP comprises a heavy chain variable region VH comprising the amino acidsequence of SEQ ID NO: 9 and a light chain variable region comprising anamino acid sequence of SEQ ID NO: 10 or the antigen binding domaincapable of specific binding to FAP comprises a heavy chain variableregion VH comprising an amino acid sequence of SEQ ID NO:17 and a lightchain variable region comprising an amino acid sequence of SEQ ID NO:18.

Bispecific Antigen Binding Molecules Binding to CD40 and FAP

In another aspect, provided is a bispecific antigen binding molecule,comprising

(i) at least one antigen binding domain capable of specific binding toCD40, comprising a heavy chain variable region (V_(H)CD40) comprising anamino acid sequence selected from the group consisting of SEQ ID NO:25,SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49,SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 andSEQ ID NO:55, and a light chain variable region (V_(L)CD40) comprisingan amino acid sequence selected from the group consisting of SEQ IDNO:26, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ IDNO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64, and

(ii) at least one antigen binding domain capable of specific binding toFAP, comprising a heavy chain variable region (V_(H)FAP) comprising anamino acid sequence of SEQ ID NO:9 and a light chain variable region(V_(L)FAP) comprising an amino acid sequence of SEQ ID NO:10, or a heavychain variable region (V_(H)FAP) comprising an amino acid sequence ofSEQ ID NO:17 and a light chain variable region (V_(L)FAP) comprising anamino acid sequence of SEQ ID NO:18.

In a further aspect, provided is a bispecific antigen binding molecule,comprising

(i) at least one antigen binding domain capable of specific binding toCD40, comprising a heavy chain variable region (V_(H)CD40) comprising anamino acid sequence selected from the group consisting of SEQ ID NO:171,SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:179, SEQ IDNO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183 and SEQ ID NO:184,and a light chain variable region (V_(L)CD40) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:175, SEQ IDNO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:185, SEQ ID NO:186, SEQID NO:187 and SEQ ID NO:188, and

(ii) at least one antigen binding domain capable of specific binding toFAP, comprising a heavy chain variable region (V_(H)FAP) comprising anamino acid sequence of SEQ ID NO:9 and a light chain variable region(V_(L)FAP) comprising an amino acid sequence of SEQ ID NO:10, or a heavychain variable region (V_(H)FAP) comprising an amino acid sequence ofSEQ ID NO:17 and a light chain variable region (V_(L)FAP) comprising anamino acid sequence of SEQ ID NO:18.

In a particular aspect, provided is a bispecific antigen bindingmolecule, comprising

(i) at least one antigen binding domain capable of specific binding toCD40, comprising a heavy chain variable region (V_(H)CD40) comprising anamino acid sequence of SEQ ID NO:171 and a light chain variable region(V_(L)CD40) comprising an amino acid sequence of SEQ ID NO:175, and

(ii) at least one antigen binding domain capable of specific binding toFAP, comprising a heavy chain variable region (V_(H)FAP) comprising anamino acid sequence of SEQ ID NO:9 and a light chain variable region(V_(L)FAP) comprising an amino acid sequence of SEQ ID NO:10, or a heavychain variable region (V_(H)FAP) comprising an amino acid sequence ofSEQ ID NO:17 and a light chain variable region (V_(L)FAP) comprising anamino acid sequence of SEQ ID NO:18.

In another particular aspect, provided is a bispecific antigen bindingmolecule, comprising

(i) at least one antigen binding domain capable of specific binding toCD40, comprising a heavy chain variable region (V_(H)CD40) comprising anamino acid of SEQ ID NO:179 or SEQ ID

NO:182 and a light chain variable region (V_(L)CD40) comprising an aminoacid sequence of SEQ ID NO:185, and

(ii) at least one antigen binding domain capable of specific binding toFAP, comprising a heavy chain variable region (V_(H)FAP) comprising anamino acid sequence of SEQ ID NO:9 and a light chain variable region(V_(L)FAP) comprising an amino acid sequence of SEQ ID NO:10, or a heavychain variable region (V_(H)FAP) comprising an amino acid sequence ofSEQ ID NO:17 and a light chain variable region (V_(L)FAP) comprising anamino acid sequence of SEQ ID NO:18.

In a further aspect, provided is a bispecific antigen binding molecule,wherein

-   (i) the antigen binding domain capable of specific binding to CD40    comprises a heavy chain variable region (V_(H)CD40) comprising the    amino acid sequence of SEQ ID NO: 25 and a light chain variable    region (V_(L)CD40) comprising an amino acid sequence of SEQ ID NO:    26 and-   (ii) the antigen binding domain capable of specific binding to FAP    comprises a heavy chain variable region VH comprising an amino acid    sequence of SEQ ID NO:9 and a light chain variable region comprising    an amino acid sequence of SEQ ID NO:10.

Furthermore, provided is a bispecific antigen binding molecule, wherein

-   (i) the antigen binding domain capable of specific binding to CD40    comprises a heavy chain variable region (V_(H)CD40) comprising the    amino acid sequence of SEQ ID NO: 25 and a light chain variable    region (V_(L)CD40) comprising an amino acid sequence of SEQ ID NO:    26 and-   (ii) the antigen binding domain capable of specific binding to FAP    comprises a heavy chain variable region VH comprising an amino acid    sequence of SEQ ID NO:17 and a light chain variable region    comprising an amino acid sequence of SEQ ID NO:18.

In another aspect, provided is a bispecific antigen binding molecule,wherein

-   (i) the antigen binding domain capable of specific binding to CD40    comprises a heavy chain variable region (V_(H)CD40) comprising the    amino acid sequence of SEQ ID NO: 47 and a light chain variable    region (V_(L)CD40) comprising an amino acid sequence of SEQ ID NO:    57 and-   (ii) the antigen binding domain capable of specific binding to FAP    comprises a heavy chain variable region VH comprising an amino acid    sequence of SEQ ID NO:17 and a light chain variable region    comprising an amino acid sequence of SEQ ID NO:18.

In another aspect, provided is a bispecific antigen binding molecule,wherein

-   (i) the antigen binding domain capable of specific binding to CD40    comprises a heavy chain variable region (V_(H)CD40) comprising an    amino acid sequence selected from the group consisting of SEQ ID    NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:179,    SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183 and SEQ    ID NO:184, and a light chain variable region (V_(L)CD40) comprising    an amino acid sequence selected from the group consisting of SEQ ID    NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:185,    SEQ ID NO:186, SEQ ID NO:187 and SEQ ID NO:188, and-   (ii) the antigen binding domain capable of specific binding to FAP    comprises a heavy chain variable region VH comprising an amino acid    sequence of SEQ ID NO:17 and a light chain variable region    comprising an amino acid sequence of SEQ ID NO:18.

Bispecific, Monovalent Antigen Binding Molecules (1+1 format)

In one aspect, the invention relates to bispecifc antigen bindingmolecules comprising (a) one antigen binding domain capable of specificbinding to a CD40, (b) one antigen binding domain capable of specificbinding to a target cell antigen, and (c) a Fc domain composed of afirst and a second subunit capable of stable association.

In a particular aspect, provided is a bispecific antigen bindingmolecule, wherein said molecule comprises (a) a first Fab fragmentcapable of specific binding to CD40, (b) a second Fab fragment capableof specific binding to a target cell antigen, and (c) a Fc domaincomposed of a first and a second subunit capable of stable association.In one aspect, the target cell antigen is FAP.

In one aspect, provided is a bispecific antigen binding molecule,wherein said molecule comprises

-   (i) a first Fab fragment capable of specific binding to CD40,    comprising a heavy chain variable region (V_(H)CD40) comprising an    amino acid sequence selected from the group consisting of SEQ ID    NO:25, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ    ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53,    SEQ ID NO:54 and SEQ ID NO:55, and a light chain variable region    (V_(L)CD40) comprising an amino acid sequence selected from the    group consisting of SEQ ID NO:26, SEQ ID NO:56, SEQ ID NO:57, SEQ ID    NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ    ID NO:63 and SEQ ID NO:64, and-   (ii) a second Fab fragment capable of specific binding to FAP,    comprising a heavy chain variable region (V_(H)FAP) comprising an    amino acid sequence of SEQ ID NO:9 and a light chain variable region    (V_(L)FAP) comprising an amino acid sequence of SEQ ID NO:10, or a    heavy chain variable region (V_(H)FAP) comprising an amino acid    sequence of SEQ ID NO:17 and a light chain variable region    (V_(L)FAP) comprising an amino acid sequence of SEQ ID NO:18.

In one aspect, provided is a bispecific antigen binding moleculecomprising a first heavy chain (HC1) comprising the amino acid sequenceof SEQ ID NO:141, a second heavy chain (HC2) comprising the amino acidsequence of SEQ ID NO:140, a first light chain comprising the amino acidsequence of SEQ ID NO:138 and a second light chain comprising the aminoacid sequence of SEQ ID NO:137.

In another aspect, provided is a bispecific antigen binding molecule,wherein said molecule comprises

-   (i) a first Fab fragment capable of specific binding to CD40,    comprising a heavy chain variable region (V_(H)CD40) comprising an    amino acid sequence selected from the group consisting of SEQ ID    NO:25, SEQ ID NO:33, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173,    SEQ ID NO:174, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID    NO:182, SEQ ID NO:183 and SEQ ID NO:184, and a light chain variable    region (V_(L)CD40) comprising an amino acid sequence selected from    the group consisting of SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:175,    SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:185, SEQ ID    NO:186, SEQ ID NO:187 and SEQ ID NO:188, and-   (ii) a second Fab fragment capable of specific binding to FAP,    comprising a heavy chain variable region (V_(H)FAP) comprising an    amino acid sequence of SEQ ID NO:9 and a light chain variable region    (V_(L)FAP) comprising an amino acid sequence of SEQ ID NO:10, or a    heavy chain variable region (V_(H)FAP) comprising an amino acid    sequence of SEQ ID NO:17 and a light chain variable region    (V_(L)FAP) comprising an amino acid sequence of SEQ ID NO:18.

In one particular aspect, provided is a bispecific antigen bindingmolecule, wherein said molecule comprises

-   (i) a first Fab fragment capable of specific binding to CD40,    comprising a heavy chain variable region (V_(H)CD40) comprising an    amino acid sequence selected from the group consisting of SEQ ID    NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:179,    SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183 and SEQ    ID NO:184, and a light chain variable region (V_(L)CD40) comprising    an amino acid sequence selected from the group consisting of SEQ ID    NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:185,    SEQ ID NO:186, SEQ ID NO:187 and SEQ ID NO:188, and-   (ii) a second Fab fragment capable of specific binding to FAP,    comprising a heavy chain variable region (V_(H)FAP) comprising an    amino acid sequence of SEQ ID NO:9 and a light chain variable region    (V_(L)FAP) comprising an amino acid sequence of SEQ ID NO:10, or a    heavy chain variable region (V_(H)FAP) comprising an amino acid    sequence of SEQ ID NO:17 and a light chain variable region    (V_(L)FAP) comprising an amino acid sequence of SEQ ID NO:18.

In a particular aspect, provided is a bispecific antigen bindingmolecule, wherein said molecule comprises (i) a first Fab fragmentcapable of specific binding to CD40, comprising a heavy chain variableregion (V_(H)CD40) comprising an amino acid sequence of SEQ ID NO:171and a light chain variable region (V_(L)CD40) comprising an amino acidsequence of SEQ ID NO:175, and (ii) a second Fab fragment capable ofspecific binding to FAP, comprising a heavy chain variable region(V_(H)FAP) comprising an amino acid sequence of SEQ ID NO:17 and a lightchain variable region (V_(L)FAP) comprising an amino acid sequence ofSEQ ID NO:18.

In another particular aspect, provided is a bispecific antigen bindingmolecule, wherein said molecule comprises (i) a first Fab fragmentcapable of specific binding to CD40, comprising a heavy chain variableregion (V_(H)CD40) comprising an amino acid sequence of SEQ ID NO:179 orSEQ ID NO:182, and a light chain variable region (V_(L)CD40) comprisingan amino acid sequence of SEQ ID NO:185, and (ii) a second Fab fragmentcapable of specific binding to FAP, comprising a heavy chain variableregion (V_(H)FAP) comprising an amino acid sequence of SEQ ID NO:17 anda light chain variable region (V_(L)FAP) comprising an amino acidsequence of SEQ ID NO:18.

In a particular aspect, provided is a bispecific antigen bindingmolecule comprising a first heavy chain (HC1) comprising the amino acidsequence of SEQ ID NO:163, a second heavy chain (HC2) comprising theamino acid sequence of SEQ ID NO:164, a first light chain comprising theamino acid sequence of SEQ ID NO:165 and a second light chain comprisingthe amino acid sequence of SEQ ID NO:162.

Bispecific Antigen Binding Molecules Bivalent for Binding to CD40 andMonovalent for Binding to the Target Cell Antigen (2+1 Format)

In another aspect, the invention provides a bispecific antigen bindingmolecule comprising

-   (a) two antigen binding domains capable of specific binding to CD40,-   (b) one antigen binding domain capable of specific binding to the    target cell antigen, and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association.

Thus, provided is a bispecific antigen binding molecule, wherein thebispecific antigen binding molecule is bivalent for CD40 and monovalentfor the target cell antigen.

In one aspect, the bispecific antigen binding molecule comprises

-   (a) two light chains and two heavy chains of an antibody comprising    two Fab fragments capable of specific binding to CD40 and the Fc    domain, and-   (b) a VH and VL domain capable of specific binding to a target cell    antigen, wherein the VH domain and the VL domain are each connected    via a peptide linker to one of the C-termini of the two heavy    chains.

In a particular aspect, the peptide linker comprises an amino acidsequence selected from SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:152 andSEQ ID NO:153. More particularly, the peptide linker comprises the SEQID NO:153.

In a particular aspect, the bispecific antigen binding moleculecomprises

-   (a) two light chains and two heavy chains of an antibody comprising    two Fab fragments capable of specific binding to CD40 and the Fc    domain, and-   (b) a VH and VL domain capable of specific binding to a target cell    antigen, wherein the VH domain is connected via a peptide linker to    the C-terminus of one of the heavy chains and wherein the VL domain    is connected via a peptide linker to the C-terminus of the second    heavy chain.

In another particular aspect, the bispecific antigen binding moleculecomprises

-   (a) two light chains and two heavy chains of an antibody comprising    two Fab fragments capable of specific binding to CD40 and the Fc    domain, and-   (b) a VH and VL domain capable of specific binding to a target cell    antigen, wherein the VH domain is connected via a peptide linker to    the C-terminus of the Fc knob heavy chain and wherein the VL domain    is connected via a peptide linker to the C-terminus of the Fc hole    heavy chain.

In one aspect, the bispecific antigen binding molecule comprises

-   (a) two light chains and two heavy chains of an antibody comprising    two Fab fragments capable of specific binding to CD40 and the Fc    domain, and-   (b) a VH and VL domain capable of specific binding to a target cell    antigen, wherein the VL domain is connected via a peptide linker to    the C-terminus of the Fc knob heavy chain and wherein the VH domain    is connected via a peptide linker to the C-terminus of the Fc hole    heavy chain.

In one aspect, the invention relates to a bispecific antigen bindingmolecule, comprising

-   (a) two Fab fragments capable of specific binding to CD40 connected    to a Fc region, and-   (b) one antigen binding domain capable of specific binding to FAP    connected to the C-terminus of the Fc region.

In a particular aspect, the invention provides a bispecific antigenbinding molecule comprising

-   (a) two light chains and two heavy chains of an antibody comprising    two Fab fragments capable of specific binding to CD40, and a Fc    region, and-   (b) a VH and a VL of an antigen binding domain capable specific    binding to FAP, wherein the

VH is connected to the C-terminus of one of the two heavy chains of (a),and wherein the VL is connected to the C-terminus of the other of thetwo heavy chains of (a).

In another aspect, the invention relates to a bispecific antigen bindingmolecule, comprising

-   (a) two heavy chains, each heavy chain comprising a VH and CH1    domain of a Fab fragment capable of specific binding to CD40 and a    Fc region subunit,-   (b) two light chains, each light chain comprising a VL and CL domain    of a Fab fragment capable of specific binding to CD40, and-   (c) a VH and a VL of an antigen binding domain capable of specific    binding to FAP, wherein the VH is connected to the C-terminus of one    of the two heavy chains of (a), and wherein the VL is connected to    the C-terminus of the other of the two heavy chains of (a)

In particular, the VH domain is a heavy chain variable region (V_(H)FAP)comprising an amino acid sequence of SEQ ID NO:9 or of SEQ ID NO:17 andthe VL domain is a light chain variable region (V_(L)FAP) comprising anamino acid sequence of SEQ ID NO:10 or of SEQ ID NO:18. Moreparticularly, the VH domain is a heavy chain variable region (V_(L)FAP)comprising an amino acid sequence of SEQ ID NO:17 and the VL domain is alight chain variable region (V_(L)FAP) comprising an amino acid sequenceof SEQ ID NO:18.

In a particular aspect, the invention provides a bispecific antigenbinding molecule comprising

-   (a) two light chains, each comprising the amino acid sequence of SEQ    ID NO:82, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:88, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:89, or-   (b) two light chains, each comprising the amino acid sequence of SEQ    ID NO:133, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:134, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:135.

In another particular aspect, the bispecific antigen binding moleculecomprises

-   (a) two light chains and two heavy chains of an antibody comprising    two Fab fragments capable of specific binding to CD40 and the Fc    domain, and-   (b) a Fab fragment capable of specific binding to a target cell    antigen, wherein the Fab fragment is connected via a peptide linker    to the C-terminus of one of the heavy chains.

In one aspect, provided is a bispecific antigen binding molecule,wherein the bispecific antigen binding molecule comprises

-   (a) two heavy chains, each heavy chain comprising a VH and CH1    domain of a Fab fragment capable of specific binding to CD40, and a    Fc region subunit,-   (b) two light chains, each light chain comprising a VL and CL domain    of a Fab fragment capable of specific binding to CD40, and-   (c) one Fab fragment capable of specific binding to FAP, wherein the    Fab fragment is connected to the C-terminus of one of the two heavy    chains of (a).

In particular, the Fab fragment capable of specific binding is acrossover fab fragment.

In one aspect, the invention provides a bispecific antigen bindingmolecule comprising two light chains, each comprising the amino acidsequence of SEQ ID NO:137, one light chain comprising the amino acidsequence of SEQ ID NO:138, a first heavy chain comprising the amino acidsequence of SEQ ID NO:139, and a second heavy chain comprising the aminoacid sequence of SEQ ID NO:136.

In a further aspect, provided is a bispecific antigen binding molecule,comprising

(a) two heavy chains, each heavy chain comprising a VH and CH1 domain ofa Fab fragment capable of specific binding to CD40 and a Fc regionsubunit,

(b) two light chains, each light chain comprising a VL and CL domain ofa Fab fragment capable of specific binding to CD40, and

(c) a crossover fab fragment capable of specific binding to FAPcomprising a VL-CH1 chain and a VH-CL chain, wherein the VH-CL chain isconnected to the C-terminus of one of the two heavy chains of (a).

In one aspect, the VH-CL chain is connected to the C-terminus of the FCknob heavy chain.

In another aspect, provided is a bispecific antigen binding molecule,comprising

(a) two heavy chains, each heavy chain comprising a VH and CH1 domain ofa Fab fragment capable of specific binding to CD40 and a Fc regionsubunit,

(b) two light chains, each light chain comprising a VL and CL domain ofa Fab fragment capable of specific binding to CD40, and

(c) a crossover fab fragment capable of specific binding to FAPcomprising a VL-CH1 chain and a VH-CL chain, wherein the VL-CH1 chain isconnected to the C-terminus of one of the two heavy chains of (a).

In one aspect, the VL-CH1 chain is connected to the C-terminus of the FCknob heavy chain.

In a particular aspect, the invention provides a bispecific antigenbinding molecule comprising

-   (a) two light chains, each comprising the amino acid sequence of SEQ    ID NO:165, one light chain comprising the amino acid sequence of SEQ    ID NO:162, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:167, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:168, or-   (b) two light chains, each comprising the amino acid sequence of SEQ    ID NO:248, one light chain comprising the amino acid sequence of SEQ    ID NO:162, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:251, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:252, or-   (c) two light chains, each comprising the amino acid sequence of SEQ    ID NO:248, one light chain comprising the amino acid sequence of SEQ    ID NO:138, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:253, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:252, or-   (d) two light chains, each comprising the amino acid sequence of SEQ    ID NO:248, one light chain comprising the amino acid sequence of SEQ    ID NO:254, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:255, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:252, or-   (e) two light chains, each comprising the amino acid sequence of SEQ    ID NO:256, one light chain comprising the amino acid sequence of SEQ    ID NO:254, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:257, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:258.

Bispecific Antigen Binding Molecules in Head-to-Tail Format (2+1)

In another aspect, provided is a bispecific antigen binding molecule,comprising

(a) a heavy chain comprising a VH and CH1 domain of a Fab fragmentcapable of specific binding to CD40 and a Fc region subunit,

(b) a heavy chain comprising a VH and CH1 domain of a Fab fragmentcapable of specific binding to CD40, a VL and CH1 domain of a Fabfragment capable of specific binding to FAP and a Fc region subunit,

(c) two light chains, each light chain comprising a VL and CL domain ofa Fab fragment capable of specific binding to CD40, and

(d) a light chain comprising a VH and CL domain of a Fab fragmentcapable of specific binding to FAP.

In particular, provided is a bispecific antigen binding moleculecomprising a first heavy chain comprising the amino acid sequence of SEQID NO:164, a second heavy chain comprising the amino acid sequence ofSEQ ID NO:166, two light chains each comprising the amino acid sequenceof SEQ ID NO:165 and a light chain comprising the amino acid sequence ofSEQ ID NO:162.

Bispecific Antigen Binding Molecules Bivalent for Binding to CD40 andBivalent for Binding to the Target Cell Antigen (2+2 Format)

In another aspect, the invention provides a bispecific antigen bindingmolecule comprising

-   (a) two antigen binding domains capable of specific binding to CD40,-   (b) two antigen binding domains capable of specific binding to the    target cell antigen, and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association.

Thus, provided is a bispecific antigen binding molecule, wherein thebispecific antigen binding molecule is bivalent for CD40 and bivalentfor the target cell antigen.

In one aspect, provided is a bispecific antigen binding molecule,wherein the bispecific antigen binding molecule comprises

(a) two heavy chains, each heavy chain comprising a VH and CH1 domain ofa Fab fragment capable of specific binding to CD40, and a Fc regionsubunit,

(b) two light chains, each light chain comprising a VL and CL domain ofa Fab fragment capable of specific binding to CD40, and

(c) two Fab fragments capable of specific binding to FAP, wherein one ofthe Fab fragments is connected to the C-terminus of one of the two heavychains of (a), and the other of the Fab fragments is connected to theC-terminus of the other of the two heavy chains of (a).

In a particular aspect, the invention provides a bispecific antigenbinding molecule comprising

-   (a) two light chains, each comprising the amino acid sequence of SEQ    ID NO:86, two light chains, each comprising the amino acid sequence    of SEQ ID NO:87, and two heavy chains, each comprising the amino    acid sequence of SEQ ID NO:90, or-   (b) two light chains, each comprising the amino acid sequence of SEQ    ID NO:137, two light chains, each comprising the amino acid sequence    of SEQ ID NO:138, and two heavy chains, each comprising the amino    acid sequence of SEQ ID NO:136.

Bispecific Antigen Binding Molecules Tetravalent for Binding to CD40 andMonovalent for Binding to the Target Cell Antigen (4+1 Format)

In another aspect, the invention provides a bispecific antigen bindingmolecule comprising

-   (a) four antigen binding domains capable of specific binding to    CD40,-   (b) one antigen binding domain capable of specific binding to a    target cell antigen, and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association:

Thus, provided is a bispecific antigen binding molecule, wherein thebispecific antigen binding molecule is tetravalent for CD40 andmonovalent for the target cell antigen.

In one aspect, provided is a bispecific antigen binding molecule,wherein the four antigen binding domains capable of specific binding toCD40 are Fab fragments and each two thereof are fused to each other atthe heavy chain, optionally via a peptide linker. In a particularaspect, the peptide linker comprises the amino acid sequence of SEQ IDNO:148. More particularly, the antigen binding molecule comprises twoheavy chains comprising each a VHCH1-peptide linker-VHCH1 fragment. In aparticular aspect, the peptide linker has the amino acid sequence of SEQID NO:148.

In another aspect, a bispecific antigen binding molecule is provided,wherein the antigen binding domain capable of specific binding to atarget cell antigen comprises a VH and VL domain and wherein the VHdomain is connected via a peptide linker to the C-terminus of the firstsubunit of the Fc domain and the VL domain is connected via a peptidelinker to the C-terminus of the second subunit of the Fc domain.

In a particular aspect, the bispecific antigen binding moleculecomprises

(a) four light chains, each light chain comprising a VL and CL domain ofa Fab fragment capable of specific binding to CD40,

(b) two heavy chains, wherein each of the heavy chain comprises a VH andCH1 domain of a Fab fragment capable of specific binding to CD40 fusedto a VH and CH1 domain of a second Fab fragment capable of specificbinding to CD40, and a Fc region subunit, and

(c) a VH and VL domain capable of specific binding to a target cellantigen, wherein the VH domain is connected via a peptide linker to theC-terminus of one of the heavy chains and wherein the VL domain isconnected via a peptide linker to the C-terminus of the second heavychain.

In a particular aspect, the peptide linker comprises an amino acidsequence selected from SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:152 andSEQ ID NO:153. More particularly, the peptide linker comprises the SEQID NO:153.

In one aspect, provided is a bispecific antigen binding moleculecomprising

(a) four light chains, each light chain comprising a light chainvariable region (V_(L)CD40) comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:175,SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:185, SEQ IDNO:186, SEQ ID NO:187 and SEQ ID NO:188,

(b) two heavy chains, wherein each of the heavy chain comprisesVH-CH1-VH-CH1 and a Fc region subunit, and wherein both VH domainscomprise a heavy chain variable region (V_(H)CD40) comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:25, SEQ IDNO:33, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183and SEQ ID NO:184, and

(c) a VH and VL domain capable of specific binding to a target cellantigen, wherein the VH domain is connected via a peptide linker to theC-terminus of one of the heavy chains and wherein the VL domain isconnected via a peptide linker to the C-terminus of the second heavychain.

In one aspect, the invention relates to a bispecific antigen bindingmolecule, comprising

-   (a) four Fab fragments capable of specific binding to CD40, (b) a VH    and a VL domain capable of specific binding to FAP, and (c) a Fc    domain composed of a first and a second subunit capable of stable    association.

In particular, the VH domain is a heavy chain variable region (V_(H)FAP)comprising an amino acid sequence of SEQ ID NO:9 or of SEQ ID NO:17 andthe VL domain is a light chain variable region (V_(L)FAP) comprising anamino acid sequence of SEQ ID NO:10 or of SEQ ID NO:18. Moreparticularly, the VH domain is a heavy chain variable region (V_(H)FAP)comprising an amino acid sequence of SEQ ID NO:17 and the VL domain is alight chain variable region (V_(L)FAP) comprising an amino acid sequenceof SEQ ID NO:18.

In a particular aspect, the invention provides a bispecific antigenbinding molecule comprising

-   (a) four light chains, each comprising the amino acid sequence of    SEQ ID NO:82, a first heavy chain comprising the amino acid sequence    of SEQ ID NO:83, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:84, or-   (b) four light chains, each comprising the amino acid sequence of    SEQ ID NO:133, a first heavy chain comprising the amino acid    sequence of SEQ ID NO:131, and a second heavy chain comprising the    amino acid sequence of SEQ ID NO:132.

In another particular aspect, the invention provides a bispecificantigen binding molecule capable of specific binding to murine CD40comprising

-   (a) four light chains, each comprising the amino acid sequence of    SEQ ID NO:97, a first heavy chain comprising the amino acid sequence    of SEQ ID NO:95, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:96.

In one aspect, provided is a bispecific antigen binding molecule,wherein the bispecific antigen binding molecule comprises

(a) four light chains, each light chain comprising a VL and CL domain ofa Fab fragment capable of specific binding to CD40,

(b) two heavy chains, wherein each of the heavy chain comprises a VH andCH1 domain of a Fab fragment capable of specific binding to CD40 fusedto a VH and CH1 domain of a second Fab fragment capable of specificbinding to CD40, and a Fc region subunit, and

-   (c) one Fab fragment capable of specific binding to FAP, wherein the    Fab fragment is connected to the C-terminus of one of the two heavy    chains of (b).

In particular, the Fab fragment capable of specific binding is acrossover fab fragment.

In one aspect, provided is a bispecific antigen binding molecule,wherein the bispecific antigen binding molecule comprises

(a) two heavy chains, each heavy chain comprising a VH and CH1 domain ofa Fab fragment capable of specific binding to CD40, and a Fc regionsubunit,

(b) two light chains, each light chain comprising a VL and CL domain ofa Fab fragment capable of specific binding to CD40, and

(c) one Fab fragment capable of specific binding to FAP, wherein the Fabfragment is connected to the C-terminus of one of the two heavy chainsof (a).

In particular, the Fab fragment capable of specific binding is acrossover fab fragment.

In a further aspect, provided is a bispecific antigen binding molecule,comprising

(a) four light chains, each light chain comprising a VL and CL domain ofa Fab fragment capable of specific binding to CD40, wherein the VLcomprises a light chain variable region (V_(L)CD40) comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:26, SEQ IDNO:34, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQID NO:185, SEQ ID NO:186, SEQ ID NO:187 and SEQ ID NO:188, and

(b) two heavy chains, each heavy chain comprising a VH-CH1-VH-CH1 chainand a Fc region subunit, wherein both VH domains comprise a heavy chainvariable region (V_(H)CD40) comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:171,SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:179, SEQ IDNO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183 and SEQ ID NO:184,and

(c) a crossover Fab fragment capable of specific binding to FAPcomprising a VL-CH1 chain and a VH-CL chain, wherein the VH-CL chain isconnected to the C-terminus of one of the two heavy chains of (b).

In one aspect, the VH-CL chain is connected to the C-terminus of the FCknob heavy chain.

In a further aspect, provided is a bispecific antigen binding molecule,comprising

(a) four light chains, each light chain comprising a VL and CL domain ofa Fab fragment capable of specific binding to CD40, wherein the VLcomprises a light chain variable region (V_(L)CD40) comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:26, SEQ IDNO:34, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQID NO:185, SEQ ID NO:186, SEQ ID NO:187 and SEQ ID NO:188, and

(b) two heavy chains, each heavy chain comprising a VH-CH1-VH-CH1 chainand a Fc region subunit, wherein both VH domains comprise a heavy chainvariable region (V_(H)CD40) comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:171,SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:179, SEQ IDNO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183 and SEQ ID NO:184,and

(c) a crossover fab fragment capable of specific binding to FAPcomprising a VL-CH1 chain and a VH-CL chain, wherein the VL-CH1 chain isconnected to the C-terminus of one of the two heavy chains of (b).

In one aspect, the VL-CH1 chain is connected to the C-terminus of the FCknob heavy chain.

In a particular aspect, the invention provides a bispecific antigenbinding molecule comprising

-   (a) four light chains, each comprising the amino acid sequence of    SEQ ID NO:165, one light chain comprising the amino acid sequence of    SEQ ID NO:162, a first heavy chain comprising the amino acid    sequence of SEQ ID NO:169, and a second heavy chain comprising the    amino acid sequence of SEQ ID NO:170, or-   (b) two light chains, each comprising the amino acid sequence of SEQ    ID NO:243, one light chain comprising the amino acid sequence of SEQ    ID NO:162, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:244, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:245, or-   (c) two light chains, each comprising the amino acid sequence of SEQ    ID NO:243, one light chain comprising the amino acid sequence of SEQ    ID NO:162, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:246, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:247, or-   (d) two light chains, each comprising the amino acid sequence of SEQ    ID NO:248, one light chain comprising the amino acid sequence of SEQ    ID NO:162, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:249, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:250.

Bispecific Antigen Binding Molecules Tetravalent for Binding to CD40 andBivalent for Binding to the Target Cell Antigen (4+2 format)

In another aspect, the invention provides a bispecific antigen bindingmolecule comprising

-   (a) four antigen binding domains capable of specific binding to    CD40,-   (b) two antigen binding domains capable of specific binding to a    target cell antigen, and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association:

Thus, provided is a bispecific antigen binding molecule, wherein thebispecific antigen binding molecule is tetravalent for CD40 and bivalentfor the target cell antigen.

In one aspect, provided is a bispecific antigen binding molecule,wherein the four antigen binding domains capable of specific binding toCD40 are Fab fragments and each two thereof are fused to each other,optionally via a peptide linker. In a particular aspect, the peptidelinker comprises the amino acid sequence of SEQ ID NO:148. Moreparticularly, the antigen binding molecule comprises two heavy chainscomprising each a VHCH1-peptide linker-VHCH1 fragment. In a particularaspect, the peptide linker has the amino acid sequence of SEQ ID NO:148.

In another aspect, a bispecific antigen binding molecule is provided,wherein the antigen binding domains capable of specific binding to atarget cell antigen are Fab fragments and wherein the first Fab fragmentis connected via a peptide linker to the C-terminus of the first subunitof the Fc domain and the second Fab fragment is connected via a peptidelinker to the C-terminus of the second subunit of the Fc domain. In oneaspect, the two Fab fragments capable of specific binding to the targetcell antigen are crossover Fab fragments each comprising a VL-CH1 chainand a VH-CL chain, and wherein the VL-CH1 chain is connected to theC-terminus of one of the two heavy chains.

In a particular aspect, the invention provides a bispecific antigenbinding molecule comprising four light chains, each comprising the aminoacid sequence of SEQ ID NO:86, two light chains, each comprising theamino acid sequence of SEQ ID NO:87, and two heavy chains comprising theamino acid sequence of SEQ ID NO:85.

In another aspect, the invention provides a bispecific antigen bindingmolecule capable of specific binding to murine CD40 comprising fourlight chains, each comprising the amino acid sequence of SEQ ID NO:100,two light chains, each comprising the amino acid sequence of SEQ IDNO:99, and two heavy chains comprising the amino acid sequence of SEQ IDNO:98.

Fc Domain Modifications Reducing Fc Receptor Binding and/or EffectorFunction

The bispecific antigen binding molecules of the invention furthercomprise a Fc domain composed of a first and a second subunit capable ofstable association.

In certain aspects, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

The Fc domain confers favorable pharmacokinetic properties to thebispecific antibodies of the invention, including a long serum half-lifewhich contributes to good accumulation in the target tissue and afavorable tissue-blood distribution ratio. At the same time it may,however, lead to undesirable targeting of the bispecific antibodies ofthe invention to cells expressing Fc receptors rather than to thepreferred antigen-bearing cells. Accordingly, in particular embodimentsthe Fc domain of the bispecific antibodies of the invention exhibitsreduced binding affinity to an Fc receptor and/or reduced effectorfunction, as compared to a native IgG Fc domain, in particular an IgG1Fc domain or an IgG4 Fc domain. More particularly, the Fc domain is anIgG1 Fc domain.

In one such aspect the Fc domain (or the bispecific antigen bindingmolecule of the invention comprising said Fc domain) exhibits less than50%, preferably less than 20%, more preferably less than 10% and mostpreferably less than 5% of the binding affinity to an Fc receptor, ascompared to a native IgG1 Fc domain (or the bispecific antigen bindingmolecule of the invention comprising a native IgG1 Fc domain), and/orless than 50%, preferably less than 20%, more preferably less than 10%and most preferably less than 5% of the effector function, as comparedto a native IgG1 Fc domain (or the bispecific antigen binding moleculeof the invention comprising a native IgG1 Fc domain). In one aspect, theFc domain (or the bispecific antigen binding molecule of the inventioncomprising said Fc domain) does not substantially bind to an Fc receptorand/or induce effector function. In a particular aspect the Fc receptoris an Fey receptor. In one aspect, the Fc receptor is a human Fcreceptor. In one aspect, the Fc receptor is an activating Fc receptor.In a specific aspect, the Fc receptor is an activating human Fcγreceptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, mostspecifically human FcγRIIIa. In one aspect, the Fc receptor is aninhibitory Fc receptor. In a specific aspect, the Fc receptor is aninhibitory human Fey receptor, more specifically human FcγRIIB In oneaspect the effector function is one or more of CDC, ADCC, ADCP, andcytokine secretion. In a particular aspect, the effector function isADCC. In one aspect, the Fc domain domain exhibits substantially similarbinding affinity to neonatal Fc receptor (FcRn), as compared to a nativeIgG1 Fc domain. Substantially similar binding to FcRn is achieved whenthe Fc domain (or the the bispecific antigen binding molecule of theinvention comprising said Fc domain) exhibits greater than about 70%,particularly greater than about 80%, more particularly greater thanabout 90% of the binding affinity of a native IgG1 Fc domain (or the thebispecific antigen binding molecule of the invention comprising a nativeIgG1 Fc domain) to FcRn.

In a particular aspect, the Fc domain is engineered to have reducedbinding affinity to an Fc receptor and/or reduced effector function, ascompared to a non-engineered Fc domain. In a particular aspect, the Fcdomain of the bispecific antigen binding molecule of the inventioncomprises one or more amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function.Typically, the same one or more amino acid mutation is present in eachof the two subunits of the Fc domain. In one aspect, the amino acidmutation reduces the binding affinity of the Fc domain to an Fcreceptor. In another aspect, the amino acid mutation reduces the bindingaffinity of the Fc domain to an Fc receptor by at least 2-fold, at least5-fold, or at least 10-fold. In one aspect, the bispecific antigenbinding molecule of the invention comprising an engineered Fc domainexhibits less than 20%, particularly less than 10%, more particularlyless than 5% of the binding affinity to an Fc receptor as compared tobispecific antibodies of the invention comprising a non-engineered Fcdomain. In a particular aspect, the Fc receptor is an Fcγ receptor. Inother aspects, the Fc receptor is a human Fc receptor. In one aspect,the Fc receptor is an inhibitory Fc receptor. In a specific aspect, theFc receptor is an inhibitory human Fey receptor, more specifically humanFcγRIIB In some aspects the Fc receptor is an activating Fc receptor. Ina specific aspect, the Fc receptor is an activating human Fcγ receptor,more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specificallyhuman FcγRIIIa. Preferably, binding to each of these receptors isreduced. In some aspects, binding affinity to a complement component,specifically binding affinity to C1q, is also reduced. In one aspect,binding affinity to neonatal Fc receptor (FcRn) is not reduced.

Substantially similar binding to FcRn, i.e. preservation of the bindingaffinity of the Fc domain to said receptor, is achieved when the Fcdomain (or the bispecific antigen binding molecule of the inventioncomprising said Fc domain) exhibits greater than about 70% of thebinding affinity of a non-engineered form of the Fc domain (or thebispecific antigen binding molecule of the invention comprising saidnon-engineered form of the Fc domain) to FcRn. The Fc domain, or the thebispecific antigen binding molecule of the invention comprising said Fcdomain, may exhibit greater than about 80% and even greater than about90% of such affinity. In certain embodiments the Fc domain of thebispecific antigen binding molecule of the invention is engineered tohave reduced effector function, as compared to a non-engineered Fcdomain. The reduced effector function can include, but is not limitedto, one or more of the following: reduced complement dependentcytotoxicity (CDC), reduced antibody-dependent cell-mediatedcytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis(ADCP), reduced cytokine secretion, reduced immune complex-mediatedantigen uptake by antigen-presenting cells, reduced binding to NK cells,reduced binding to macrophages, reduced binding to monocytes, reducedbinding to polymorphonuclear cells, reduced direct signaling inducingapoptosis, reduced dendritic cell maturation, or reduced T cell priming.

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581). Certain antibody variants with improved or diminishedbinding to FcRs are described. (e.g. U.S. Pat. No. 6,737,056; WO2004/056312, and Shields, R. L. et al., J. Biol. Chem. 276 (2001)6591-6604).

In one aspect of the invention, the Fc domain comprises an amino acidsubstitution at a position of E233, L234, L235, N297, P331 and P329. Insome aspects, the Fc domain comprises the amino acid substitutions L234Aand L235A (“LALA”). In one such embodiment, the Fc domain is an IgG1 Fcdomain, particularly a human IgG1 Fc domain. In one aspect, the Fcdomain comprises an amino acid substitution at position P329. In a morespecific aspect, the amino acid substitution is P329A or P329G,particularly P329G. In one embodiment the Fc domain comprises an aminoacid substitution at position P329 and a further amino acid substitutionselected from the group consisting of E233P, L234A, L235A, L235E, N297A,N297D or P331S. In more particular embodiments the Fc domain comprisesthe amino acid mutations L234A, L235A and P329G (“P329G LALA”). The“P329G LALA” combination of amino acid substitutions almost completelyabolishes Fcγ receptor binding of a human IgG1 Fc domain, as describedin PCT Patent Application No. WO 2012/130831 Al. Said document alsodescribes methods of preparing such mutant Fc domains and methods fordetermining its properties such as Fc receptor binding or effectorfunctions. Such antibody is an IgG1 with mutations L234A and L235A orwith mutations L234A, L235A and P329G (numbering according to EU indexof Kabat et al., Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md., 1991).

In one aspect, the Fc domain is an IgG4 Fc domain. In a more specificembodiment, the Fc domain is an IgG4 Fc domain comprising an amino acidsubstitution at position 5228 (Kabat numbering), particularly the aminoacid substitution S228P. In a more specific embodiment, the Fc domain isan IgG4 Fc domain comprising amino acid substitutions L235E and S228Pand P329G. This amino acid substitution reduces in vivo Fab arm exchangeof IgG4 antibodies (see Stubenrauch et al., Drug Metabolism andDisposition 38, 84-91 (2010)).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer, R. L. et al., J. Immunol. 117 (1976)587-593, and Kim, J. K. et al., J. Immunol. 24 (1994) 2429-2434), aredescribed in US 2005/0014934. Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).See also Duncan, A. R. and Winter, G., Nature 322 (1988) 738-740; U.S.Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerningother examples of Fc region variants.

Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBlAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. A suitable such binding assay isdescribed herein. Alternatively, binding affinity of Fc domains or cellactivating bispecific antigen binding molecules comprising an Fc domainfor Fc receptors may be evaluated using cell lines known to expressparticular Fc receptors, such as human NK cells expressing FcγIIIareceptor. Effector function of an Fc domain, or bispecific antigenbinding molecules of the invention comprising an Fc domain, can bemeasured by methods known in the art. A suitable assay for measuringADCC is described herein. Other examples of in vitro assays to assessADCC activity of a molecule of interest are described in U.S. Pat. No.5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986)and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S.Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987).Alternatively, non-radioactive assays methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g. in a animal model such as that disclosed in Clynes et al.,Proc Natl Acad Sci USA 95, 652-656 (1998).

The following section describes preferred aspects of the bispecificantigen binding molecules of the invention comprising Fc domainmodifications reducing Fc receptor binding and/or effector function. Inone aspect, the invention relates to the bispecific antigen bindingmolecule (a) at least one antigen binding domain capable of specificbinding to CD40, (b) at least one antigen binding domain capable ofspecific binding to a target cell antigen, and (c) a Fc domain composedof a first and a second subunit capable of stable association, whereinthe Fc domain comprises one or more amino acid substitution that reducesthe binding affinity of the antibody to an Fc receptor, in particulartowards Fcγ receptor. In another aspect, the invention relates to thebispecific antigen binding molecule comprising (a) at least one antigenbinding domain capable of specific binding to CD40, (b) at least oneantigen binding domain capable of specific binding to a target cellantigen, and (c) a Fc domain composed of a first and a second subunitcapable of stable association, wherein the Fc domain comprises one ormore amino acid substitution that reduces effector function. Inparticular aspect, the Fc domain is of human IgG1 subclass with theamino acid mutations L234A, L235A and P329G (numbering according toKabat EU index).

Fc Domain Modifications Promoting Heterodimerization

The bispecific antigen binding molecules of the invention comprisedifferent antigen-binding sites, fused to one or the other of the twosubunits of the Fc domain, thus the two subunits of the Fc domain may becomprised in two non-identical polypeptide chains. Recombinantco-expression of these polypeptides and subsequent dimerization leads toseveral possible combinations of the two polypeptides. To improve theyield and purity of the bispecific antigen binding molecules of theinvention in recombinant production, it will thus be advantageous tointroduce in the Fc domain of the bispecific antigen binding moleculesof the invention a modification promoting the association of the desiredpolypeptides.

Accordingly, in particular aspects the invention relates to thebispecific antigen binding molecule comprising (a) at least one antigenbinding domain capable of specific binding to CD40, (b) at least oneantigen binding domain capable of specific binding to a target cellantigen, and (c) a Fc domain composed of a first and a second subunitcapable of stable association, wherein the Fc domain comprises amodification promoting the association of the first and second subunitof the Fc domain. The site of most extensive protein-protein interactionbetween the two subunits of a human IgG Fc domain is in the CH3 domainof the Fc domain. Thus, in one aspect said modification is in the CH3domain of the Fc domain.

In a specific aspect said modification is a so-called “knob-into-hole”modification, comprising a “knob” modification in one of the twosubunits of the Fc domain and a “hole” modification in the other one ofthe two subunits of the Fc domain. Thus, the invention relates to thebispecific antigen binding molecule comprising (a) at least one antigenbinding domain capable of specific binding to CD40, (b) at least oneantigen binding domain capable of specific binding to a target cellantigen, and (c) a Fc domain composed of a first and a second subunitcapable of stable association, wherein the first subunit of the Fcdomain comprises knobs and the second subunit of the Fc domain comprisesholes according to the knobs into holes method. In a particular aspect,the first subunit of the Fc domain comprises the amino acidsubstitutions S354C and T366W (EU numbering) and the second subunit ofthe Fc domain comprises the amino acid substitutions Y349C, T366S andY407V (numbering according to Kabat EU index).

The knob-into-hole technology is described e.g. in U.S. Pat. No.5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621(1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, themethod involves introducing a protuberance (“knob”) at the interface ofa first polypeptide and a corresponding cavity (“hole”) in the interfaceof a second polypeptide, such that the protuberance can be positioned inthe cavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine).

Accordingly, in one aspect, in the CH3 domain of the first subunit ofthe Fc domain of the bispecific antigen binding molecules of theinvention an amino acid residue is replaced with an amino acid residuehaving a larger side chain volume, thereby generating a protuberancewithin the CH3 domain of the first subunit which is positionable in acavity within the CH3 domain of the second subunit, and in the CH3domain of the second subunit of the Fc domain an amino acid residue isreplaced with an amino acid residue having a smaller side chain volume,thereby generating a cavity within the CH3 domain of the second subunitwithin which the protuberance within the CH3 domain of the first subunitis positionable. The protuberance and cavity can be made by altering thenucleic acid encoding the polypeptides, e.g. by site-specificmutagenesis, or by peptide synthesis. In a specific aspect, in the CH3domain of the first subunit of the Fc domain the threonine residue atposition 366 is replaced with a tryptophan residue (T366W), and in theCH3 domain of the second subunit of the Fc domain the tyrosine residueat position 407 is replaced with a valine residue (Y407V). In oneaspect, in the second subunit of the Fc domain additionally thethreonine residue at position 366 is replaced with a serine residue(T366S) and the leucine residue at position 368 is replaced with analanine residue (L368A).

In yet a further aspect, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C), and in the second subunit of the Fc domainadditionally the tyrosine residue at position 349 is replaced by acysteine residue (Y349C). Introduction of these two cysteine residuesresults in formation of a disulfide bridge between the two subunits ofthe Fc domain, further stabilizing the dimer (Carter (2001), J ImmunolMethods 248, 7-15). In a particular aspect, the first subunit of the Fcdomain comprises the amino acid substitutions S354C and T366W (EUnumbering) and the second subunit of the Fc domain comprises the aminoacid substitutions Y349C, T366S and Y407V (numbering according to KabatEU index).

In an alternative aspect, a modification promoting association of thefirst and the second subunit of the Fc domain comprises a modificationmediating electrostatic steering effects, e.g. as described in PCTpublication WO 2009/089004. Generally, this method involves replacementof one or more amino acid residues at the interface of the two Fc domainsubunits by charged amino acid residues so that homodimer formationbecomes electrostatically unfavorable but heterodimerizationelectrostatically favorable.

The C-terminus of the heavy chain of the bispecific antibody as reportedherein can be a complete C-terminus ending with the amino acid residuesPGK. The C-terminus of the heavy chain can be a shortened C-terminus inwhich one or two of the C terminal amino acid residues have beenremoved. In one preferred aspect, the C-terminus of the heavy chain is ashortened C-terminus ending PG. In one aspect of all aspects as reportedherein, a bispecific antibody comprising a heavy chain including aC-terminal CH3 domain as specified herein, comprises the C-terminalglycine-lysine dipeptide (G446 and K447, numbering according to Kabat EUindex). In one embodiment of all aspects as reported herein, abispecific antibody comprising a heavy chain including a C-terminal CH3domain, as specified herein, comprises a C-terminal glycine residue(G446, numbering according to Kabat EU index).

Modifications in the Fab Domains

In one aspect, the invention relates to a bispecific antigen bindingmolecule comprising (a) a first Fab fragment capable of specific bindingto CD40, (b) a second Fab fragment capable of specific binding to atarget cell antigen, and (c) a Fc domain composed of a first and asecond subunit capable of stable association, wherein in one of the Fabfragments either the variable domains VH and VL or the constant domainsCH1 and CL are exchanged. The bispecific antibodies are preparedaccording to the Crossmab technology.

Multispecific antibodies with a domain replacement/exchange in onebinding arm (CrossMabVH-VL or CrossMabCH-CL) are described in detail inWO2009/080252 and Schaefer, W. et al, PNAS, 108 (2011) 11187-1191. Theyclearly reduce the byproducts caused by the mismatch of a light chainagainst a first antigen with the wrong heavy chain against the secondantigen (compared to approaches without such domain exchange).

In one aspect, the invention relates to a bispecific antigen bindingmolecule comprising (a) a first Fab fragment capable of specific bindingto CD40, (b) a second Fab fragment capable of specific binding to atarget cell antigen, and (c) a Fc domain composed of a first and asecond subunit capable of stable association, wherein in one of the Fabfragments the constant domains CL and CH1 are replaced by each other sothat the CH1 domain is part of the light chain and the CL domain is partof the heavy chain. More particularly, in the second Fab fragmentcapable of specific binding to a target cell antigen the constantdomains CL and CH1 are replaced by each other so that the CH1 domain ispart of the light chain and the CL domain is part of the heavy chain.

In a particular aspect, the invention relates a bispecific antigenbinding molecule comprising (a) a first Fab fragment capable of specificbinding to CD40, (b) a second Fab fragment capable of specific bindingto a target cell antigen, wherein the constant domains CL and CH1 arereplaced by each other so that the CH1 domain is part of the light chainand the CL domain is part of the heavy chain. Such a molecule is calleda monovalent bispecific antigen binding molecule.

In another aspect, the invention relates to a bispecific antigen bindingmolecule, comprising (a) two light chains and two heavy chains of anantibody comprising two Fab fragments capable of specific binding toCD40 and the Fc domain, and (b) two additional Fab fragments capable ofspecific binding to a target cell antigen, wherein said additional Fabfragments are each connected via a peptide linker to the C-terminus ofthe heavy chains of (a). In a particular aspect, the additional Fabfragments are Fab fragments, wherein the variable domains VL and VH arereplaced by each other so that the VH domain is part of the light chainand the VL domain is part of the heavy chain.

Thus, in a particular aspect, the invention comprises a bispecificantigen binding molecule, comprising (a) two light chains and two heavychains of an antibody comprising two Fab fragments capable of specificbinding to CD40 and the Fc domain, and (b) two additional Fab fragmentscapable of specific binding to a target cell antigen, wherein said twoadditional Fab fragments capable of specific binding to a target cellantigen are crossover Fab fragments wherein the variable domains VL andVH are replaced by each other and the VL-CH chains are each connectedvia a peptide linker to the C-terminus of the heavy chains of (a).

In another aspect, and to further improve correct pairing, thebispecific antigen binding molecule comprising (a) a first Fab fragmentcapable of specific binding to CD40, (b) a second Fab fragment capableof specific binding to a target cell antigen, and (c) a Fc domaincomposed of a first and a second subunit capable of stable association,can contain different charged amino acid substitutions (so-called“charged residues”). These modifications are introduced in the crossedor non-crossed CH1 and CL domains. In a particular aspect, the inventionrelates to a bispecific antigen binding molecule, wherein in one of CLdomains the amino acid at position 123 (EU numbering) has been replacedby arginine (R) and the amino acid at position 124 (EU numbering) hasbeen substituted by lysine (K) and wherein in one of the CH1 domains theamino acids at position 147 (EU numbering) and at position 213 (EUnumbering) have been substituted by glutamic acid (E).

Exemplary Antibodies of the Invention

In one aspect, the invention provides new antibodies and antibodyfragments that specifically bind to CD40. These antibodies have superiorproperties compared to the known CD40 antibodies that make themespecially suitable for the incorporation into bispecific antigenbinding molecules comprising another antigen binding moiety capable ofspecific binding to a target cell antigen. The new antibodies arefurther characterized in that they are producable in high amounts andwith high titers, that they show high thermal stability (as measured bythe aggregation temperature T_(agg)), or in that they possess a highdegree of humanness and may therefore be less immunogenic in the humanbody. The percentage of humanness of the VH and VL sequences as comparedto the human germline sequences can be determined by the methodsdescribed in Abhinandan, K. R. and Martin, Andrew C. R. 2007, J. Mol.Biol. 2007, 369, 852-862. The corresponding data are shown in Tables 24and 25.

In one aspect, provided is an antibody that specifically binds to CD40,wherein said antibody comprises

-   -   (i) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:171 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:175,    -   (ii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:173 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:177,    -   (iii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:174 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:178,    -   (iv) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:171 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:177,    -   (v) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:171 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:178,    -   (vi) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:173 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:175, or    -   (vii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:173 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:178, or    -   (viii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:174 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:175, or    -   (viii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:174 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:177, or    -   (ix) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:171 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:176, or    -   (x) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:172 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:175, or    -   (xi) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:172 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:176, or    -   (xii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:172 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:177, or    -   (xiii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:172 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:178, or    -   (xiv) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:173 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:176, or    -   (xv) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:174 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:176.

In another aspect, provided is an antibody that competes for bindingwith an antibody that specifically binds to CD40, wherein said antibodycomprises any of the heavy chain variable region VH and a light chainvariable region VL of (i) to (xv) as defined herein before.

In one aspect, provided is an antibody that competes for binding with anantibody that specifically binds to CD40, wherein said antibodycomprises a heavy chain variable region VH comprising an amino acidsequence of SEQ ID NO:171 and a light chain variable region VLcomprising an amino acid sequence of SEQ ID NO:175.

In a further aspect, provided is an antibody that specifically binds toCD40, wherein said antibody comprises

-   -   (i) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:179 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:185,    -   (ii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:180 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:185,    -   (iii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:181 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:185,    -   (iv) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:182 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:185,    -   (v) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:179 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:186,    -   (vi) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:180 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:186, or    -   (vii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:181 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:186, or    -   (viii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:1182 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:186.

In another aspect, provided is an antibody that competes for bindingwith an antibody that specifically binds to CD40, wherein said antibodycomprises any of the heavy chain variable region VH and a light chainvariable region VL of (i) to (viii) as defined herein before.

In one aspect, provided is an antibody that competes for binding with anantibody that specifically binds to CD40, wherein said antibodycomprises a heavy chain variable region VH comprising an amino acidsequence of SEQ ID NO:179 and a light chain variable region VLcomprising an amino acid sequence of SEQ ID NO:185. In particular,provided is an antibody that specifically binds to CD40, wherein saidantibody comprises a heavy chain variable region VH comprising an aminoacid sequence of SEQ ID NO:182 and and a light chain variable region VLcomprising an amino acid sequence of SEQ ID NO:185.

Polynucleotides

The invention further provides isolated polynucleotides encoding abispecific antigen binding molecule as described herein or a fragmentthereof or polynucleotides encoding an antibody as described herein.

The isolated polynucleotides encoding bispecific antigen bindingmolecules of the invention may be expressed as a single polynucleotidethat encodes the entire antigen binding molecule or as multiple (e.g.,two or more) polynucleotides that are co-expressed. Polypeptides encodedby polynucleotides that are co-expressed may associate through, e.g.,disulfide bonds or other means to form a functional antigen bindingmolecule. For example, the light chain portion of an immunoglobulin maybe encoded by a separate polynucleotide from the heavy chain portion ofthe immunoglobulin. When co-expressed, the heavy chain polypeptides willassociate with the light chain polypeptides to form the immunoglobulin.

In some aspects, the isolated polynucleotide encodes a polypeptidecomprised in the bispecific molecule according to the invention asdescribed herein.

In one aspect, the present invention is directed to an isolatedpolynucleotide encoding a bispecific antigen binding molecule,comprising (a) at least one antigen binding domain capable of specificbinding to CD40, (b) at least one antigen binding domain capable ofspecific binding to a target cell antigen, and (c) a Fc domain composedof a first and a second subunit capable of stable association.

In certain embodiments the polynucleotide or nucleic acid is DNA. Inother embodiments, a polynucleotide of the present invention is RNA, forexample, in the form of messenger RNA (mRNA). RNA of the presentinvention may be single stranded or double stranded.

Recombinant Methods

Bispecific antigen binding molecules of the invention may be obtained,for example, by recombinant production. For recombinant production oneor more polynucleotide encoding the bispecific antigen binding moleculeor polypeptide fragments thereof are provided. The one or morepolynucleotide encoding the bispecific antigen binding molecule areisolated and inserted into one or more vectors for further cloningand/or expression in a host cell. Such polynucleotide may be readilyisolated and sequenced using conventional procedures. In one aspect ofthe invention, a vector, preferably an expression vector, comprising oneor more of the polynucleotides of the invention is provided. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing the coding sequence of thebispecific antigen binding molecule (fragment) along with appropriatetranscriptional/translational control signals. These methods include invitro recombinant DNA techniques, synthetic techniques and in vivorecombination/genetic recombination. See, for example, the techniquesdescribed in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL,Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and WileyInterscience, N.Y. (1989). The expression vector can be part of aplasmid, virus, or may be a nucleic acid fragment. The expression vectorincludes an expression cassette into which the polynucleotide encodingthe bispecific antigen binding molecule or polypeptide fragments thereof(i.e. the coding region) is cloned in operable association with apromoter and/or other transcription or translation control elements. Asused herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, if present, but any flankingsequences, for example promoters, ribosome binding sites,transcriptional terminators, introns, 5′ and 3′ untranslated regions,and the like, are not part of a coding region. Two or more codingregions can be present in a single polynucleotide construct, e.g. on asingle vector, or in separate polynucleotide constructs, e.g. onseparate (different) vectors. Furthermore, any vector may contain asingle coding region, or may comprise two or more coding regions, e.g. avector of the present invention may encode one or more polypeptides,which are post- or co-translationally separated into the final proteinsvia proteolytic cleavage. In addition, a vector, polynucleotide, ornucleic acid of the invention may encode heterologous coding regions,either fused or unfused to a polynucleotide encoding the bispecificantigen binding molecule of the invention or polypeptide fragmentsthereof, or variants or derivatives thereof. Heterologous coding regionsinclude without limitation specialized elements or motifs, such as asecretory signal peptide or a heterologous functional domain. Anoperable association is when a coding region for a gene product, e.g. apolypeptide, is associated with one or more regulatory sequences in sucha way as to place expression of the gene product under the influence orcontrol of the regulatory sequence(s). Two DNA fragments (such as apolypeptide coding region and a promoter associated therewith) are“operably associated” if induction of promoter function results in thetranscription of mRNA encoding the desired gene product and if thenature of the linkage between the two DNA fragments does not interferewith the ability of the expression regulatory sequences to direct theexpression of the gene product or interfere with the ability of the DNAtemplate to be transcribed. Thus, a promoter region would be operablyassociated with a nucleic acid encoding a polypeptide if the promoterwas capable of effecting transcription of that nucleic acid. Thepromoter may be a cell-specific promoter that directs substantialtranscription of the DNA only in predetermined cells. Othertranscription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can be operably associated with the polynucleotide to directcell-specific transcription.

Suitable promoters and other transcription control regions are disclosedherein. A variety of transcription control regions are known to thoseskilled in the art. These include, without limitation, transcriptioncontrol regions, which function in vertebrate cells, such as, but notlimited to, promoter and enhancer segments from cytomegaloviruses (e.g.the immediate early promoter, in conjunction with intron-A), simianvirus 40 (e.g. the early promoter), and retroviruses (such as, e.g. Roussarcoma virus). Other transcription control regions include thosederived from vertebrate genes such as actin, heat shock protein, bovinegrowth hormone and rabbit â-globin, as well as other sequences capableof controlling gene expression in eukaryotic cells. Additional suitabletranscription control regions include tissue-specific promoters andenhancers as well as inducible promoters (e.g. promoters inducibletetracyclins). Similarly, a variety of translation control elements areknown to those of ordinary skill in the art. These include, but are notlimited to ribosome binding sites, translation initiation andtermination codons, and elements derived from viral systems(particularly an internal ribosome entry site, or IRES, also referred toas a CITE sequence). The expression cassette may also include otherfeatures such as an origin of replication, and/or chromosome integrationelements such as retroviral long terminal repeats (LTRs), oradeno-associated viral (AAV) inverted terminal repeats (ITRs).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. For example, if secretionof the bispecific antigen binding molecule or polypeptide fragmentsthereof is desired, DNA encoding a signal sequence may be placedupstream of the nucleic acid encoding the bispecific antigen bindingmolecule of the invention or polypeptide fragments thereof. According tothe signal hypothesis, proteins secreted by mammalian cells have asignal peptide or secretory leader sequence which is cleaved from themature protein once export of the growing protein chain across the roughendoplasmic reticulum has been initiated. Those of ordinary skill in theart are aware that polypeptides secreted by vertebrate cells generallyhave a signal peptide fused to the N-terminus of the polypeptide, whichis cleaved from the translated polypeptide to produce a secreted or“mature” form of the polypeptide. In certain embodiments, the nativesignal peptide, e.g. an immunoglobulin heavy chain or light chain signalpeptide is used, or a functional derivative of that sequence thatretains the ability to direct the secretion of the polypeptide that isoperably associated with it. Alternatively, a heterologous mammaliansignal peptide, or a functional derivative thereof, may be used. Forexample, the wild-type leader sequence may be substituted with theleader sequence of human tissue plasminogen activator (TPA) or mouseβ-glucuronidase.

DNA encoding a short protein sequence that could be used to facilitatelater purification (e.g. a histidine tag) or assist in labeling thefusion protein may be included within or at the ends of thepolynucleotide encoding a bispecific antigen binding molecule of theinvention or polypeptide fragments thereof.

In a further aspect of the invention, a host cell comprising one or morepolynucleotides of the invention is provided. In certain aspects, a hostcell comprising one or more vectors of the invention is provided. Thepolynucleotides and vectors may incorporate any of the features, singlyor in combination, described herein in relation to polynucleotides andvectors, respectively. In one aspect, a host cell comprises (e.g. hasbeen transformed or transfected with) a vector comprising apolynucleotide that encodes (part of) a bispecific antigen bindingmolecule of the invention of the invention. As used herein, the term“host cell” refers to any kind of cellular system which can beengineered to generate the fusion proteins of the invention or fragmentsthereof. Host cells suitable for replicating and for supportingexpression of antigen binding molecules are well known in the art. Suchcells may be transfected or transduced as appropriate with theparticular expression vector and large quantities of vector containingcells can be grown for seeding large scale fermenters to obtainsufficient quantities of the antigen binding molecule for clinicalapplications. Suitable host cells include prokaryotic microorganisms,such as E. coli, or various eukaryotic cells, such as Chinese hamsterovary cells (CHO), insect cells, or the like. For example, polypeptidesmay be produced in bacteria in particular when glycosylation is notneeded. After expression, the polypeptide may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forpolypeptide-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized”, resulting in theproduction of a polypeptide with a partially or fully humanglycosylation pattern. See Gerngross, Nat Biotech 22, 1409-1414 (2004),and Li et al., Nat Biotech 24, 210-215 (2006).

Suitable host cells for the expression of (glycosylated) polypeptidesare also derived from multicellular organisms (invertebrates andvertebrates). Examples of invertebrate cells include plant and insectcells. Numerous baculoviral strains have been identified which may beused in conjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells. Plant cell cultures can also be utilized ashosts. See e.g. U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548,7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology forproducing antibodies in transgenic plants). Vertebrate cells may also beused as hosts. For example, mammalian cell lines that are adapted togrow in suspension may be useful. Other examples of useful mammalianhost cell lines are monkey kidney CV1 line transformed by SV40 (COS-7);human embryonic kidney line (293 or 293T cells as described, e.g., inGraham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells(BHK), mouse sertoli cells (TM4 cells as described, e.g., in Mather,Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), Africangreen monkey kidney cells (VERO-76), human cervical carcinoma cells(HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A),human lung cells (W138), human liver cells (Hep G2), mouse mammary tumorcells (MMT 060562), TRI cells (as described, e.g., in Mather et al.,Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells.Other useful mammalian host cell lines include Chinese hamster ovary(CHO) cells, including dhfr-CHO cells (Urlaub et al., Proc Natl Acad SciUSA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 andSp2/0. For a review of certain mammalian host cell lines suitable forprotein production, see, e.g., Yazaki and Wu, Methods in MolecularBiology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp.255-268 (2003). Host cells include cultured cells, e.g., mammaliancultured cells, yeast cells, insect cells, bacterial cells and plantcells, to name only a few, but also cells comprised within a transgenicanimal, transgenic plant or cultured plant or animal tissue. In oneembodiment, the host cell is a eukaryotic cell, preferably a mammaliancell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonickidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).Standard technologies are known in the art to express foreign genes inthese systems. Cells expressing a polypeptide comprising either theheavy or the light chain of an immunoglobulin, may be engineered so asto also express the other of the immunoglobulin chains such that theexpressed product is an immunoglobulin that has both a heavy and a lightchain.

In one aspect, a method of producing a bispecific antigen bindingmolecule of the invention or polypeptide fragments thereof is provided,wherein the method comprises culturing a host cell comprisingpolynucleotides encoding the bispecific antigen binding molecule of theinvention or polypeptide fragments thereof, as provided herein, underconditions suitable for expression of the bispecific antigen bindingmolecule of the invention or polypeptide fragments thereof, andrecovering the bispecific antigen binding molecule of the invention orpolypeptide fragments thereof from the host cell (or host cell culturemedium).

Bispecific molecules of the invention prepared as described herein maybe purified by art-known techniques such as high performance liquidchromatography, ion exchange chromatography, gel electrophoresis,affinity chromatography, size exclusion chromatography, and the like.The actual conditions used to purify a particular protein will depend,in part, on factors such as net charge, hydrophobicity, hydrophilicityetc., and will be apparent to those having skill in the art. Foraffinity chromatography purification an antibody, ligand, receptor orantigen can be used to which the bispecific antigen binding moleculebinds. For example, for affinity chromatography purification of fusionproteins of the invention, a matrix with protein A or protein G may beused. Sequential Protein A or G affinity chromatography and sizeexclusion chromatography can be used to isolate an antigen bindingmolecule essentially as described in the examples. The purity of thebispecific antigen binding molecule or fragments thereof can bedetermined by any of a variety of well-known analytical methodsincluding gel electrophoresis, high pressure liquid chromatography, andthe like. For example, the bispecific antigen binding moleculesexpressed as described in the Examples were shown to be intact andproperly assembled as demonstrated by reducing and non-reducingSDS-PAGE.

Assays

The antigen binding molecules provided herein may be characterized fortheir binding properties and/or biological activity by various assaysknown in the art. In particular, they are characterized by the assaysdescribed in more detail in the examples.

1. Binding Assay

Binding of the bispecific antigen binding molecule provided herein tothe corresponding target expressing cells may be evaluated for exampleby using a murine fibroblast cell line expressing human FibroblastActivation Protein (FAP) and flow cytometry (FACS) analysis. Binding ofthe bispecific antigen binding molecules provided herein to CD40 may bedetermined by using Raji cells as described in Example 4.2.8.

2. Activity Assays

Bispecific antigen binding molecules of the invention are tested forbiological activity. Biological activity may include efficacy andspecificity of the bispecific antigen binding molecules. Efficacy andspecificity are demonstrated by assays showing agonistic signalingthrough the CD40 receptor upon binding of the target antigen.Furthermore in vitro T cell priming assays are conducted using dendriticcells (DCs) that have been incubated with the bispecific antigen bindingmolecules.

Pharmaceutical Compositions, Formulations and Routes of Administation

In a further aspect, the invention provides pharmaceutical compositionscomprising any of the bispecific antigen binding molecules providedherein, e.g., for use in any of the below therapeutic methods. In oneembodiment, a pharmaceutical composition comprises any of the bispecificantigen binding molecules provided herein and at least onepharmaceutically acceptable excipient. In another embodiment, apharmaceutical composition comprises any of the bispecific antigenbinding molecules provided herein and at least one additionaltherapeutic agent, e.g., as described below.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of one or more bispecific antigenbinding molecules dissolved or dispersed in a pharmaceuticallyacceptable excipient. The phrases “pharmaceutical or pharmacologicallyacceptable” refers to molecular entities and compositions that aregenerally non-toxic to recipients at the dosages and concentrationsemployed, i.e. do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a pharmaceutical composition thatcontains at least one bispecific antigen binding molecule according tothe invention and optionally an additional active ingredient will beknown to those of skill in the art in light of the present disclosure,as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. MackPrinting Company, 1990, incorporated herein by reference. In particular,the compositions are lyophilized formulations or aqueous solutions. Asused herein, “pharmaceutically acceptable excipient” includes any andall solvents, buffers, dispersion media, coatings, surfactants,antioxidants, preservatives (e.g. antibacterial agents, antifungalagents), isotonic agents, salts, stabilizers and combinations thereof,as would be known to one of ordinary skill in the art.

Parenteral compositions include those designed for administration byinjection, e.g. subcutaneous, intradermal, intralesional, intravenous,intraarterial intramuscular, intrathecal or intraperitoneal injection.For injection, the bispecific antigen binding molecules of the inventionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiological saline buffer. The solution may contain formulatory agentssuch as suspending, stabilizing and/or dispersing agents. Alternatively,the bispecific antigen binding molecules may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. Sterile injectable solutions are prepared by incorporatingthe antigen binding molecules of the invention in the required amount inthe appropriate solvent with various of the other ingredients enumeratedbelow, as required. Sterility may be readily accomplished, e.g., byfiltration through sterile filtration membranes. Generally, dispersionsare prepared by incorporating the various sterilized active ingredientsinto a sterile vehicle which contains the basic dispersion medium and/orthe other ingredients. In the case of sterile powders for thepreparation of sterile injectable solutions, suspensions or emulsion,the preferred methods of preparation are vacuum-drying or freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filtered liquidmedium thereof. The liquid medium should be suitably buffered ifnecessary and the liquid diluent first rendered isotonic prior toinjection with sufficient saline or glucose. The composition must bestable under the conditions of manufacture and storage, and preservedagainst the contaminating action of microorganisms, such as bacteria andfungi. It will be appreciated that endotoxin contamination should bekept minimally at a safe level, for example, less than 0.5 ng/mgprotein. Suitable pharmaceutically acceptable excipients include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Aqueous injection suspensions may contain compounds whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, dextran, or the like. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions. Additionally, suspensions of the activecompounds may be prepared as appropriate oily injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl cleats ortriglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(18th Ed. Mack Printing Company, 1990). Sustained-release preparationsmay be prepared. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe polypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. In particular embodiments, prolonged absorptionof an injectable composition can be brought about by the use in thecompositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof.

Exemplary pharmaceutically acceptable excipients herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

In addition to the compositions described previously, the antigenbinding molecules may also be formulated as a depot preparation. Suchlong acting formulations may be administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the fusion proteins may be formulated withsuitable polymeric or hydrophobic materials (for example as emulsion inan acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions comprising the bispecific antigen bindingmolecules of the invention may be manufactured by means of conventionalmixing, dissolving, emulsifying, encapsulating, entrapping orlyophilizing processes. Pharmaceutical compositions may be formulated inconventional manner using one or more physiologically acceptablecarriers, diluents, excipients or auxiliaries which facilitateprocessing of the proteins into preparations that can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

The bispecific antigen binding molecules may be formulated into acomposition in a free acid or base, neutral or salt form.Pharmaceutically acceptable salts are salts that substantially retainthe biological activity of the free acid or base. These include the acidaddition salts, e.g. those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine. Pharmaceutical salts tend to be more soluble in aqueous andother protic solvents than are the corresponding free base forms.

The composition herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

Therapeutic Methods and Compositions

Any of the bispecific antigen binding molecules provided herein may beused in therapeutic methods. For use in therapeutic methods, bispecificantigen binding molecules of the invention can be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.

In one aspect, bispecific antigen binding molecules of the invention foruse as a medicament are provided.

In further aspects, bispecific antigen binding molecules of theinvention for use (i) in inducing immune stimulation by CD40⁺antigen-presenting cells (APCs), (ii) in stimulating tumor-specific Tcell response, (iii) in causing apoptosis of tumor cells, (iv) in thetreatment of cancer, (v) in delaying progression of cancer, (vi) inprolonging the survival of a patient suffering from cancer, (vii) in thetreatment of infections are provided. In a particular aspect, bispecificantigen binding molecules of the invention for use in treating adisease, in particular for use in the treatment of cancer, are provided.

In certain aspects, bispecific antigen binding molecules of theinvention for use in a method of treatment are provided. In one aspect,the invention provides a bispecific antigen binding molecule asdescribed herein for use in the treatment of a disease in an individualin need thereof. In certain aspects, the invention provides a bispecificantigen binding molecule for use in a method of treating an individualhaving a disease comprising administering to the individual atherapeutically effective amount of the bispecific antigen bindingmolecule. In certain aspects the disease to be treated is cancer. Thesubject, patient, or “individual” in need of treatment is typically amammal, more specifically a human.

In one aspect, provided is a method for i) inducing immune stimulationby CD40+ antigen-presenting cells (APCs), (ii) stimulatingtumor-specific T cell response, (iii) causing apoptosis of tumor cells,(iv) treating of cancer, (v) delaying progression of cancer, (vi)prolonging the survival of a patient suffering from cancer, or (vii)treating of infections, wherein the method comprises administering atherapeutically effective amount of the bispecific antigen bindingmolecule of the invention to an individual in need thereof.

In a further aspect, the invention provides for the use of thebispecific antigen binding molecule of the invention in the manufactureor preparation of a medicament for the treatment of a disease in anindividual in need thereof. In one aspect, the medicament is for use ina method of treating a disease comprising administering to an individualhaving the disease a therapeutically effective amount of the medicament.In certain aspects, the disease to be treated is a proliferativedisorder, particularly cancer. Examples of cancers include, but are notlimited to, bladder cancer, brain cancer, head and neck cancer,pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterinecancer, cervical cancer, endometrial cancer, esophageal cancer, coloncancer, colorectal cancer, rectal cancer, gastric cancer, prostatecancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer,and kidney cancer. Other examples of cancer include carcinoma, lymphoma(e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, andleukemia. Other cell proliferation disorders that can be treated usingthe bispecific antigen binding molecule or antibody of the inventioninclude, but are not limited to neoplasms located in the: abdomen, bone,breast, digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid),eye, head and neck, nervous system (central and peripheral), lymphaticsystem, pelvic, skin, soft tissue, spleen, thoracic region, andurogenital system. Also included are pre-cancerous conditions or lesionsand cancer metastases. In certain embodiments the cancer is chosen fromthe group consisting of renal cell cancer, skin cancer, lung cancer,colorectal cancer, breast cancer, brain cancer, head and neck cancer. Askilled artisan readily recognizes that in many cases the the bispecificantigen binding molecule or antibody of the invention may not provide acure but may provide a benefit. In some aspects, a physiological changehaving some benefit is also considered therapeutically beneficial. Thus,in some aspects, an amount of the bispecific antigen binding molecule orantibody of the invention that provides a physiological change isconsidered an “effective amount” or a “therapeutically effectiveamount”.

For the prevention or treatment of disease, the appropriate dosage of abispecific antigen binding molecule of the invention (when used alone orin combination with one or more other additional therapeutic agents)will depend on the type of disease to be treated, the route ofadministration, the body weight of the patient, the specific molecule,the severity and course of the disease, whether the bispecific antigenbinding molecule of the invention is administered for preventive ortherapeutic purposes, previous or concurrent therapeutic interventions,the patient's clinical history and response to the bispecific antigenbinding molecule, and the discretion of the attending physician. Thepractitioner responsible for administration will, in any event,determine the concentration of active ingredient(s) in a composition andappropriate dose(s) for the individual subject. Various dosing schedulesincluding but not limited to single or multiple administrations overvarious time-points, bolus administration, and pulse infusion arecontemplated herein.

The bispecific antigen binding molecule of the invention is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of the bispecific antigen bindingmolecule can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the bispecific antigen binding molecule of the invention wouldbe in the range from about 0.005 mg/kg to about 10 mg/kg. In otherexamples, a dose may also comprise from about 1 μg/kg body weight, about5 μg/kg body weight, about 10 μg/kg body weight, about 50 μg/kg bodyweight, about 100 μg/kg body weight, about 200 μg/kg body weight, about350 μg/kg body weight, about 500 μg/kg body weight, about 1 mg/kg bodyweight, about 5 mg/kg body weight, about 10 mg/kg body weight, about 50mg/kg body weight, about 100 mg/kg body weight, about 200 mg/kg bodyweight, about 350 mg/kg body weight, about 500 mg/kg body weight, toabout 1000 mg/kg body weight or more per administration, and any rangederivable therein. In examples of a derivable range from the numberslisted herein, a range of about 0.1 mg/kg body weight to about 20 mg/kgbody weight, about 5 μg/kg body weight to about 1 mg/kg body weightetc., can be administered, based on the numbers described above. Thus,one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg(or any combination thereof) may be administered to the patient. Suchdoses may be administered intermittently, e.g. every week or every threeweeks (e.g. such that the patient receives from about two to abouttwenty, or e.g. about six doses of the fusion protein). In a particularaspect, the bispecific antigen binding molecule will be administeredevery three weeks. An initial higher loading dose, followed by one ormore lower doses may be administered. However, other dosage regimens maybe useful. The progress of this therapy is easily monitored byconventional techniques and assays.

The bispecific antigen binding molecule of the invention will generallybe used in an amount effective to achieve the intended purpose. For useto treat or prevent a disease condition, the bispecific antigen bindingmolecule of the invention, or pharmaceutical compositions thereof, areadministered or applied in a therapeutically effective amount.Determination of a therapeutically effective amount is well within thecapabilities of those skilled in the art, especially in light of thedetailed disclosure provided herein. For systemic administration, atherapeutically effective dose can be estimated initially from in vitroassays, such as cell culture assays. A dose can then be formulated inanimal models to achieve a circulating concentration range that includesthe IC₅₀ as determined in cell culture. Such information can be used tomore accurately determine useful doses in humans. Initial dosages canalso be estimated from in vivo data, e.g., animal models, usingtechniques that are well known in the art. One having ordinary skill inthe art could readily optimize administration to humans based on animaldata.

Dosage amount and interval may be adjusted individually to provideplasma levels of the bispecific antigen binding molecule of theinvention which are sufficient to maintain therapeutic effect. Usualpatient dosages for administration by injection range from about 0.1 to50 mg/kg/day, typically from about 0.1 to 1 mg/kg/day. Therapeuticallyeffective plasma levels may be achieved by administering multiple doseseach day. Levels in plasma may be measured, for example, by HPLC. Incases of local administration or selective uptake, the effective localconcentration of the bispecific antigen binding molecule or antibody ofthe invention may not be related to plasma concentration. One skilled inthe art will be able to optimize therapeutically effective local dosageswithout undue experimentation.

A therapeutically effective dose of the bispecific antigen bindingmolecule of the invention described herein will generally providetherapeutic benefit without causing substantial toxicity. Toxicity andtherapeutic efficacy of a fusion protein can be determined by standardpharmaceutical procedures in cell culture or experimental animals. Cellculture assays and animal studies can be used to determine the LD₅₀ (thedose lethal to 50% of a population) and the ED₅₀ (the dosetherapeutically effective in 50% of a population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index, whichcan be expressed as the ratio LD₅₀/ED₅₀. Bispecific antigen bindingmolecules that exhibit large therapeutic indices are preferred. In oneaspect, the the bispecific antigen binding molecule or antibody of theinvention exhibits a high therapeutic index. The data obtained from cellculture assays and animal studies can be used in formulating a range ofdosages suitable for use in humans. The dosage lies preferably within arange of circulating concentrations that include the ED50 with little orno toxicity. The dosage may vary within this range depending upon avariety of factors, e.g., the dosage form employed, the route ofadministration utilized, the condition of the subject, and the like. Theexact formulation, route of administration and dosage can be chosen bythe individual physician in view of the patient's condition (see, e.g.,Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch.1, p. 1, incorporated herein by reference in its entirety).

The attending physician for patients treated with fusion proteins of theinvention would know how and when to terminate, interrupt, or adjustadministration due to toxicity, organ dysfunction, and the like.Conversely, the attending physician would also know to adjust treatmentto higher levels if the clinical response were not adequate (precludingtoxicity). The magnitude of an administered dose in the management ofthe disorder of interest will vary with the severity of the condition tobe treated, with the route of administration, and the like. The severityof the condition may, for example, be evaluated, in part, by standardprognostic evaluation methods. Further, the dose and perhaps dosefrequency will also vary according to the age, body weight, and responseof the individual patient.

Other Agents and Treatments

The bispecific antigen binding molecule of the invention may beadministered in combination with one or more other agents in therapy.For instance, the bispecific antigen binding molecule or antibody of theinvention of the invention may be co-administered with at least oneadditional therapeutic agent. The term “therapeutic agent” encompassesany agent that can be administered for treating a symptom or disease inan individual in need of such treatment. Such additional therapeuticagent may comprise any active ingredients suitable for the particularindication being treated, preferably those with complementary activitiesthat do not adversely affect each other. In certain embodiments, anadditional therapeutic agent is another anti-cancer agent, for example amicrotubule disruptor, an antimetabolite, a topoisomerase inhibitor, aDNA intercalator, an alkylating agent, a hormonal therapy, a kinaseinhibitor, a receptor antagonist, an activator of tumor cell apoptosis,or an antiangiogenic agent. In certain aspects, an additionaltherapeutic agent is an immunomodulatory agent, a cytostatic agent, aninhibitor of cell adhesion, a cytotoxic or cytostatic agent, anactivator of cell apoptosis, or an agent that increases the sensitivityof cells to apoptotic inducers.

Thus, provided are bispecific antigen binding molecules of the inventionor pharmaceutical compositions comprising them for use in the treatmentof cancer, wherein the bispecific antigen binding molecule isadministered in combination with a chemotherapeutic agent, radiationand/or other agents for use in cancer immunotherapy.

Such other agents are suitably present in combination in amounts thatare effective for the purpose intended. The effective amount of suchother agents depends on the amount of fusion protein used, the type ofdisorder or treatment, and other factors discussed above. The thebispecific antigen binding molecule or antibody of the invention aregenerally used in the same dosages and with administration routes asdescribed herein, or about from 1 to 99% of the dosages describedherein, or in any dosage and by any route that is empirically/clinicallydetermined to be appropriate.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate compositions), and separate administration, in which case,administration of the bispecific antigen binding molecule or antibody ofthe invention can occur prior to, simultaneously, and/or following,administration of the additional therapeutic agent and/or adjuvant.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper that ispierceable by a hypodermic injection needle). At least one active agentin the composition is a bispecific antigen binding molecule of theinvention.

The label or package insert indicates that the composition is used fortreating the condition of choice. Moreover, the article of manufacturemay comprise (a) a first container with a composition contained therein,wherein the composition comprises a bispecific antigen binding moleculeof the invention; and (b) a second container with a compositioncontained therein, wherein the composition comprises a further cytotoxicor otherwise therapeutic agent. The article of manufacture in thisembodiment of the invention may further comprise a package insertindicating that the compositions can be used to treat a particularcondition.

Alternatively, or additionally, the article of manufacture may furthercomprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

TABLE C (Sequences): SEQ ID NO: Name Sequence 1 hu CD40 UniProt no.P25942, version 200 MVRLPLQCVL WGCLLTAVHP EPPTACREKQ YLINSQCCSLCQPGQKLVSD CTEFTETECL PCGESEFLDT WNRETHCHQH KYCDPNLGLR VQQKGTSETDTICTCEEGWH CTSEACESCV LHRSCSPGFG VKQIATGVSD TICEPCPVGF FSNVSSAFEKCHPWTSCETK DLVVQQAGTN KTDVVCGPQD RLRALVVIPI IFGILFAILL VLVFIKKVAKKPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 2 hu FAPUniProt no. Q12884, version 168 MKTWVKIVFG VATSAVLALL VMCIVLRPSRVHNSEENTMR ALTLKDILNG TFSYKTFFPN WISGQEYLHQ SADNNIVLYN IETGQSYTILSNRTMKSVNA SNYGLSPDRQ FVYLESDYSK LWRYSYTATY YIYDLSNGEF VRGNELPRPIQYLCWSPVGS KLAYVYQNNI YLKQRPGDPP FQITFNGREN KIFNGIPDWV YEEEMLATKYALWWSPNGKF LAYAEFNDTD IPVIAYSYYG DEQYPRTINI PYPKAGAKNP VVRIFIIDTTYPAYVGPQEV PVPAMIASSD YYFSWLTWVT DERVCLQWLK RVQNVSVLSI CDFREDWQTWDCPKTQEHIE ESRTGWAGGF FVSTPVFSYD AISYYKIFSD KDGYKHIHYI KDTVENAIQITSGKWEAINI FRVTQDSLFY SSNEFEEYPG RRNIYRISIG SYPPSKKCVT CHLRKERCQYYTASFSDYAK YYALVCYGPG IPISTLHDGR TDQEIKILEE NKELENALKN IQLPKEEIKKLEVDEITLWY KMILPPQFDR SKKYPLLIQV YGGPCSQSVR SVFAVNWISY LASKEGMVIALVDGRGTAFQ GDKLLYAVYR KLGVYEVEDQ ITAVRKFIEM GFIDEKRIAI WGWSYGGYVSSLALASGTGL FKCGIAVAPV SSWEYYASVY TERFMGLPTK DDNLEHYKNS TVMARAEYFRNVDYLLIHGT ADDNVHFQNS AQIAKALVNA QVDFQAMWYS DQNHGLSGLS TNHLYTHMTHFLKQCFSLSD 3 FAP (28H1) CDR-H1 SHAMS 4 FAP (28H1) CDR-H2AIWASGEQYYADSVKG 5 FAP (28H1) CDR-H3 GWLGNFDY 6 FAP (28H1) CDR-L1RASQSVSRSYLA 7 FAP (28H1) CDR-L2 GASTRAT 8 FAP (28H1) CDR-L3 QQGQVIPPT 9FAP(28H1) VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSS 10 FAP(28H1) VL EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIK 11 FAP(4B9) CDR-H1 SYAMS 12 FAP(4B9) CDR-H2 AIIGSGASTYYADSVKG 13FAP(4B9) CDR-H3 GWFGGFNY 14 FAP(4B9) CDR-L1 RASQSVTSSYLA 15 FAP(4B9)CDR-L2 VGSRRAT 16 FAP(4B9) CDR-L3 QQGIMLPPT 17 FAP(4B9) VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMS WVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFN YWGQGTLVTVSS 18 FAP(4B9) VLEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLA WYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKV EIK 19 hu CD40 CDR-H1 GYYIH 20 huCD40 CDR-H2 RVIPNAGGTSYNQKFKG 21 hu CD40 CDR-H3 EGIYW 22 hu CD40 CDR-L1RSSQSLVHSNGNTFLH 23 hu CD40 CDR-L2 TVSNRFS 24 hu CD40 CDR-L3 SQTTHVPWT25 hu CD40 VH EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGKGLEWVARVIPNAGGTSYNQKFKGRFTL SVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSS 26 hu CD40 VL DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQKPGKAPKLLIYTVSNRFSGVPSRFSGS GSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGQGTKVEIK 27 mu CD40 CDR-H1 DYYMA 28 mu CD40 CDR-H2 SISYDGSSTYYRDSVKG 29mu CD40 CDR-H3 HSSYFDY 30 mu CD40 CDR-L1 ASDSVSTLMH 31 mu CD40 CDR-L2LASHLES 32 mu CD40 CDR-L3 QQSWNDPWT 33 mu CD40 VHEVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMA WVRQAPTKGLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCGRHSSYFDY WGQGVMVTVSS 34 mu CD40 VLDTVLTQSPALAVSPGERVTISCRASDSVSTLMHWY QQKPGQQPKLLIYLASHLESGVPARFSGSGSGTDFTLTIDPVEADDTATYYCQQSWNDPWTFGGGTKLEL K 35 hu CD40 CDR-H1 long GYSFTGYYIH36 hu CD40 CDR-H2 (hVH_1, RVIPNNGGTSYNQKFKG hVH_2) 37 hu CD40 CDR-H2(hVH_3) RVIPNAGGTSYNQKFKG 38 hu CD40 CDR-H2 (hVH_4) RVIPQAGGTSYNQKFKG 39hu CD40 CDR-H2 (hVH_5) RVIPNNGGTSYNQKFQG 40 hu CD40 CDR-H2 (hVH_6)RVIPNNGGTSYAQKFKG 41 hu CD40 CDR-H2 (hVH_7) RVIPNNGGTSYAQKFQG 42 hu CD40CDR-H2 RVIPNAGGTSYNQKFQG (hVH_5_N288A) 43 hu CD40 CDR-H2RVIPNAGGTSYAQKFKG (hVH_6_N288A) 44 hu CD40 CDR-H2 RVIPNAGGTSYAQKFQG(hVH_7_N288A) 45 hu CD40 hVH_1 see Table 14 46 hu CD40 hVH_2 see Table14 47 hu CD40 hVH_3 see Table 14 48 hu CD40 hVH_4 see Table 14 49 huCD40 hVH_5 see Table 14 50 hu CD40 hVH_6 see Table 14 51 hu CD40 hVH_7see Table 14 52 hu CD40 hVH_2_N288A see Table 14 53 hu CD40 hVH_5_N288Asee Table 14 54 hu CD40 hVH_6_N288A see Table 14 55 hu CD40 hVH_7_N288Asee Table 14 56 hu CD40 hVK_1 see Table 15 57 hu CD40 hVK_2 see Table 1558 hu CD40 hVK_3 see Table 15 59 hu CD40 hVK_4 see Table 15 60 hu CD40hVK_5 see Table 15 61 hu CD40 hVK_6 see Table 15 62 hu CD40 hVK_7 seeTable 15 63 hu CD40 hVK_8 see Table 15 64 hu CD40 hVK_9 see Table 15 65DP47-CDR H1 SYAMS 66 DP47-CDR H2 AISGSGGSTYYADSVKG 67 DP47-CDR H3 GSGFDY68 DP47-CDR L1 RASQSVSSSYLA 69 DP47-CDR L2 GASSRAT 70 DP47-CDR L3QQYGSSPLT 71 DP47 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSS 72 DP47 VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIK 73 hu CD40 VH (nucleotide see Table 2 sequence) 74 hu CD40 VL(nucleotide see Table 2 sequence) 75 FAP 28H1 VH (nucleotide see Table 2sequence) 76 FAP 28H1 VL (nucleotide see Table 2 sequence) 77 DP47 VH(nucleotide see Table 2 sequence) 78 DP47 VL(nucleotide see Table 2sequence) 79 mu CD40 VH (nucleotide see Table 2 sequence) 80 mu CD40 VL(nucleotide see Table 2 sequence) 81 CD40 IgG heavy chain see Table 3 82CD40 light chain see Table 3 83 CD40 VHCH1-CD40 see Table 3VHCH1-Fcknob_PGLALA- 28H1 VH 84 CD40 VHCH1-CD40 see Table 3VHCH1-Fchole_PGLALA- 28H1 VL 85 CD40 VHCH1-CD40 see Table 3VHCH1-Fc_PGLALA- 28H1 VLCH1 EE 86 CD40 light chain RK see Table 3 8728H1 VHCL see Table 3 88 huCD40_Fchole_PGLALA_28H1 see Table 3 VL 89huCD40_Fcknob_PGLALA_28H1 see Table 3 VH 90 huCD40- see Table 3Fc_PGLALA_28H1_VLCH1 EE 91 CD40 VHCH1-CD40 see Table 3VHCH1-Fcknob_PGLALA- DP47 VH 92 CD40 VHCH1-CD40 see Table 3VHCH1-Fchole_PGLALA- DP47 VL 93 CD40 VHCH1-CD40 see Table 3VHCH1-Fc_PGLALA- DP47 VLCH1 EE 94 DP47VHCL see Table 3 95 muCD40VHCH1-muCD40 see Table 3 VHCH1-FcKK_DAPG- 28H1 VH 96 muCD40 VHCH1-muCD40see Table 3 VHCH1-FcDD_DAPG- 28H1 VL 97 mu CD40 light chain see Table 398 muCD40 VHCH1-muCD40 see Table 3 VHCH1-Fc_DAPG-28H1 VLCH1 99 28H1 VHCL(mu) see Table 3 100 mu CD40 light chain; ‘RK’ see Table 3 101 muCD40VHCH1-muCD40 see Table 3 VHCH1-FcKK_DAPG- DP47 VH 102 muCD40 VHCH1-muCD40 see Table 3 VHCH1-FcDD_DAPG- DP47 VL 103 muCD40 VHCH1-muCD40 seeTable 3 VHCH1-Fc_DAPG-28H1 VLCH1 104 DP47 VHCL (mu) see Table 3 105 CD40IgG heavy chain see Table 4 (nucleotide sequence) 106 CD40 light chain(nucleotide see Table 4 sequence) 107 CD40 VHCH1-CD40 see Table 4VHCH1-Fcknob_PGLALA- 28H1 VH 108 CD40 VHCH1-CD40 see Table 4VHCH1-Fchole_PGLALA- 28H1 VL 109 CD40 VHCH1-CD40 see Table 4VHCH1-Fc_PGLALA- 28H1 VLCH1 EE 110 CD40 light chain RK see Table 4 11128H1 VHCL see Table 4 112 huCD40_Fchole_PGLALA_28H1 see Table 4 VL 113huCD40_Fcknob_PGLALA_28H1 see Table 4 VH 114 huCD40- see Table 4Fc_PGLALA_28H1_VLCH1 EE 115 CD40 VHCH1-CD40 see Table 4VHCH1-Fcknob_PGLALA- DP47 VH 116 CD40 VHCH1-CD40 see Table 4VHCH1-Fchole_PGLALA- DP47 VL 117 CD40 VHCH1-CD40 see Table 4VHCH1-Fc_PGLALA- DP47 VLCH1 EE 118 DP47 VHCL see Table 4 119 muCD40VHCH1-muCD40 see Table 4 VHCH1-FcKK_DAPG- 28H1 VH 120 muCD40VHCH1-muCD40 see Table 4 VHCH1-FcDD_DAPG- 28H1 VL 121 mu CD40 lightchain see Table 4 122 muCD40 VHCH1-muCD40 see Table 4 VHCH1-Fc_DAPG-28H1VLCH1 123 28H1 VHCL (mu) see Table 4 124 mu CD40 light chain; ‘RK’ seeTable 4 125 muCD40 VHCH1-muCD40 see Table 4 VHCH1-FcKK_DAPG- DP47 VH 126muCD40 VHCH1-mu CD40 see Table 4 VHCH1-FcDD_DAPG- DP47 VL 127 muCD40VHCH1-muCD40 see Table 4 VHCH1-Fc_DAPG-28H1 VLCH1 128 DP47 VHCL (mu) seeTable 4 129 CD40 (S2C6) VH EVQLQQSGPD LVKPGASVKI SCKASGYSFT GYYIHWVKQSHGKSLEWIGR VIPNNGGTSY NQKFKGKAIL TVDKSSSTAY MELRSLTSED SAVYYCAREGIYWWGHGTTL TVSS 130 CD40 (S2C6) VL DVVVTQTPLS LPVSLGAQAS ISCRSSQSLVHSNGNTFLHW YLQKPGQSPK LLIYTVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGVYFCSQTTHVP WTFGGGTKLE IQ 131 hVH3_CD40 VHCH1- see Table 27 VHCH1-Fcknob_PGLALA- 4B9 VH 132 hVH3_CD40 VHCH1- see Table 27 VHCH1-Fchole_PGLALA- 4B9 VL 133 hVK2_CD40 light chain see Table 27 134 hVH3_CD40VHCH1-Fcknob_PGLALA- see Table 27 4B9 VH 135 hVH3_CD40VHCH1-Fchole_PGLALA- see Table 27 4B9 VL 136 hVH3_CD40-Fcknob_PGLALA_4B9_VLCH1 see Table 27 ‘EE’ 137 hVK2_CD40 LC ,RK’ see Table27 138 4B9 VHCL see Table 27 139 hVH3_CD40-Fchole_PGLALA see Table 27‘EE’ 140 hVH3_CD40-Fchole_PGLALA see Table 27 ‘EE’ 141 4B9-Fcknob_PGLALA see Table 27 142 hu FAP ectodomain + poly-RPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNW lys-tag + his₆-tagISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMK SVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVY QNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAY SYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCL QWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKH IHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKE RCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEI TLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLY AVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYY ASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQA MWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSDGKKKKKKGHHHHHH 143 mouse FAP UniProt no. P97321 144 Murine FAPRPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNW ectodomain + poly-lys-ISEQEYLHQSEDDNIVFYNIETRESYIILSNSTMK tag + his₆-tagSVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYY IYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGRENRIFNGIPDWVY EEEMLATKYALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTY PHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEE SRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAIYIFRVTQDSLFYS SNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRT DQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKS VFAVNWITYLASKEGIVIALVDGRGTAFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIW GWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRN VDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGILSGRSQNHLYTHMTHFLKQCFSLSD GKKKKKKGHHHHHH 145 Cynomolgus FAPRPPRVHNSEENTMRALTLKDILNGTFSYKTFFPNW ectodomain + poly-lys-ISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMK tag + his₆-tagSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYY IYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVY EEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPFVRIFIIDTTY PAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEE SRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYS SNEFEDYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRT DQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRS VFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIW GWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRN VDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSDG KKKKKKGHHHHHH 146 murine CD40UniProt P27512, version 160 MVSLPRLCAL WGCLLTAVHL GQCVTCSDKQ YLHDGQCCDLCQPGSRLTSH CTALEKTQCH PCDSGEFSAQ WNREIRCHQH RHCEPNQGLR VKKEGTAESDTVCTCKEGQH CTSKDCEACA QHTPCIPGFG VMEMATETTD TVCHPCPVGF FSNQSSLFEKCYPWTSCEDK NLEVLQKGTS QTNVICGLKS RMRALLVIPV VMGILITIFG VFLYIKKVVKKPKDNEILPP AARRQDPQEM EDYPGHNTAA PVQETLHGCQ PVTQEDGKES RISVQERQVTDSIALRPLV 147 Peptide linker (G4S) GGGGS 148 Peptide linker (G4S)₂GGGGSGGGGS 149 Peptide linker (SG4)₂ SGGGGSGGGG 150 Peptide linkerG4(SG4)₂ GGGGSGGGGSGGGG 151 peptide linker GSPGSSSSGS 152 (G4S)₃ peptidelinker GGGGSGGGGSGGGGS₃ 153 (G4S)₄ peptide linker GGGGSGGGGSGGGGSGGGGS154 peptide linker GSGSGSGS 155 peptide linker GSGSGNGS 156 peptidelinker GGSGSGSG 157 peptide linker GGSGSG 158 peptide linker GGSG 159peptide linker GGSGNGSG 160 peptide linker GGNGSGSG 161 peptide linkerGGNGSG 162 28H1 light chain cross see Table 6 VHCL 163 28H1 (VLCH1)_Fcknob_PGLALA see Table 6 164 CD40 (VHCH1 charged)_Fchole_PGLALA see Table6 165 CD40 light chain (charged) see Table 6 166 CD40 (VHCH1 see Table 6charged)_28H1 (VLCH1)_FC knob_PGLALA 167 CD40 (VHCH1charged)_Fcknob_PGLALA_28H1 see Table 6 (VLCH1) 168 CD40 (VHCH1charged)_Fchole_PGLALA see Table 6 169 CD40 (VHCH1 see Table 6charged_CD40 (VHCH1 charged)-Fcknob_PGLALA_28H1 (VLCH1) 170 CD40 (VHCH1see Table 6 charged_CD40 (VHCH1 charged)-Fc hole_PGLALA 171 VH1a (CD40)see Table 20 172 VH1b (CD40) see Table 20 173 VH1c (CD40) see Table 20174 VH1d (CD40) see Table 20 175 VL1a (CD40) see Table 20 176 VL1b(CD40) see Table 20 177 VL1c (CD40) see Table 20 178 VL1d (CD40) seeTable 20 179 VH2a (CD40) see Table 21 180 VH2b (CD40) see Table 21 181VH2c (CD40) see Table 21 182 VH2d (CD40) see Table 21 183 VH2ab (CD40)see Table 21 184 VH2ac (CD40) see Table 21 185 VL2a (CD40) see Table 21186 VL2b (CD40) see Table 21 187 VL2ab (CD40) see Table 21 188 VL2ac(CD40) see Table 21 189 P1AE0400 Heavy chain see Table 23 190 P1AE0400light chain see Table 23 191 P1AE0401 heavy chain see Table 23 192P1AE0401 light chain see Table 23 193 P1AE0402 heavy chain see Table 23194 P1AE0402 light chain see Table 23 195 P1AE0403 heavy chain see Table23 196 P1AE0403 light chain see Table 23 197 P1AE0404 heavy chain seeTable 23 198 P1AE0404 light chain see Table 23 199 P1AE0405 heavy chainsee Table 23 200 P1AE0405 light chain see Table 23 201 P1AE0406 heavychain see Table 23 202 P1AE0406 light chain see Table 23 203 P1AE0407heavy chain see Table 23 204 P1AE0407 light chain see Table 23 205P1AE0817 heavy chain see Table 23 206 P1AE0817 light chain see Table 23207 P1AE0818 heavy chain see Table 23 208 P1AE0818 light chain see Table23 209 P1AE0819 heavy chain see Table 23 210 P1AE0819 light chain seeTable 23 211 P1AE0993 heavy chain see Table 23 212 P1AE0993 light chainsee Table 23 213 P1AE0996 heavy chain see Table 23 214 P1AE0996 lightchain see Table 23 215 P1AE0997 heavy chain see Table 23 216 P1AE0997light chain see Table 23 217 P1AE0998 heavy chain see Table 23 218P1AE0998 light chain see Table 23 219 P1AE0999 heavy chain see Table 23220 P1AE0999 light chain see Table 23 221 P1AE1000 heavy chain see Table23 222 P1AE1000 light chain see Table 23 223 P1AE1001 heavy chain seeTable 23 224 P1AE1001 light chain see Table 23 225 P1AE1002 heavy chainsee Table 23 226 P1AE1002 light chain see Table 23 227 P1AE1003 heavychain see Table 23 228 P1AE1003 light chain see Table 23 229 P1AE1004heavy chain see Table 23 230 P1AE1004 light chain see Table 23 231P1AE1005 heavy chain see Table 23 232 P1AE1005 light chain see Table 23233 P1AE1006 heavy chain see Table 23 234 P1AE1006 light chain see Table23 235 P1AE1007 heavy chain see Table 23 236 P1AE1007 light chain seeTable 23 237 P1AE1125 heavy chain see Table 23 238 P1AE1125 light chainsee Table 23 239 P1AE1126 heavy chain see Table 23 240 P1AE1126 lightchain see Table 23 241 P1AE1135 heavy chain see Table 23 242 P1AE1135light chain see Table 23 243 VL2a (CD40) light chain see Table 28(charged) 244 VH2a (CD40) (VHCH1 see Table 28 charged_VH2a (CD40) (VHCH1charged)-Fcknob_PGLALA_28H1 (VLCH1) 245 VH2a (CD40) (VHCH1 see Table 28charged_VH2a (CD40) (VHCH1 charged)-Fchole_PGLALA 246 VH2d (CD40) (VHCH1see Table 28 charged_VH2d (CD40) (VHCH1 charged)-Fcknob_PGLALA_28H1(VLCH1) 247 VH2d (CD40) (VHCH1 see Table 28 charged_VH2d (CD40) (VHCH1charged)-Fchole_PGLALA 248 VL1a (CD40) light chain see Table 28(charged) 249 VH1a (CD40) (VHCH1)_VH1a see Table 28 (CD40) (VHCH1)Fcknob_PGLALA_28H1 (VLCH1) (charged) 250 VH1a (CD40) (VHCH1)_VH1a seeTable 28 (CD40) (VHCH1)- Fc hole_PGLALA (charged) 251 VH1a (CD40)(VHCH1) Fcknob_PGLALA_28H1 see Table 28 (VLCH1) (charged) 252 VH1a(CD40) (VHCH1) Fchole_PGLALA see Table 28 (charged) 253 VH1a (CD40)(VHCH1) Fcknob_PGLALA_4B9 see Table 28 (VLCH1) (charged) 254 4B9 lightchain cross VLCH see Table 28 255 VH1a (CD40) (VHCH1) Fcknob_PGLALA_4B9see Table 28 (VHCL) (charged) 256 VL1a (CD40) light chain see Table 28257 VH1a (CD40) (VHCH1) Fcknob_PGLALA_4B9 see Table 28 (VHCL) 258 VH1a(CD40) (VHCH1) Fchole_PGLALA see Table 28 259 P1AE0816 heavy chain seeTable 23 (control) 260 P1AE0816 light chain see Table 23 (control) 261hu CD40 CDR-H1 (VH2ab) GYYMH 262 hu CD40 CDR-H2 (VH2ab)RVIPNAGGTSYNQKFKG 263 hu CD40 CDR-H2 (VH2ac) RVIPNAGGTSYNQKVKG 264 huCD40 CDR-L1 (VL2ab) RASQSLVHSNGNTFLH 265 hu CD40 CDR-L1 (VL2ac)RSSQSIVHSNGNTFLH 266 Hu_CD40_ECD_His_AviEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFT ETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSC SPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRGG GGSHHHHHHGSGLNDIFEAQKIEWHE 267cyno_CD40_ECD_His_Avi EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCSESEFLDTWNRETRCHQHKYCDPNLGLR VQQKGTSETDTICTCEEGLHCTSESCESCVPHRSCLPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEK CRPWTSCETKDLVVQQAGTNKTDVVCGPQDRQRGGGGSHHHHHHGSGLNDIFEAQKIEWHE 268 Selicrelumab IgG2 heavyQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMH chainWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTM (control)TRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYC TNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTRFVVSVLTVVH QDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 269 Selicrelumab IgG2 lightDIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAW chainYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTD (control)FTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

-   The following numbered paragraphs (paras) describe aspects of the    present invention according to the first priority application:

1. A bispecific antigen binding molecule, comprising

-   (a) at least one antigen binding domain capable of specific binding    to CD40, and-   (b) at least one antigen binding domain capable of specific binding    to a target cell antigen.

2. The bispecific antigen binding molecule of para 1, additionallycomprising

-   (c) a Fc region composed of a first and a second subunit capable of    stable association.

3. The bispecific antigen binding molecule of para 1 or para 2, whereinthe antigen binding domain capable of specific binding to CD40 binds toa polypeptide comprising, or consisting of, the amino acid sequence ofSEQ ID NO:1.

4. The bispecific antigen binding molecule of any one of paras 1 to 3,wherein the antigen binding domain capable of specific binding to atarget cell antigen is an antigen binding domain capable of specificbinding to Fibroblast Activation Protein (FAP).

5. The bispecific antigen binding molecule of para 1 or para 2, whereinthe antigen binding domain capable of specific binding to FAP binds to apolypeptide comprising, or consisting of, the amino acid sequence of SEQID NO:2.

6. The bispecific antigen binding molecule of any one of paras 1 to 5,wherein the antigen binding domain capable of specific binding to FAPcomprises

(a) a heavy chain variable region (V_(H)FAP) comprising (i) CDR-H1comprising the amino acid sequence of SEQ ID NO:3, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:4, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:5, and a light chainvariable region (V_(L)FAP) comprising (iv) CDR-L1 comprising the aminoacid sequence of SEQ ID NO:6, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO:7, and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO:8, or

-   (b) a heavy chain variable region (V_(H)FAP) comprising (i) CDR-H1    comprising the amino acid sequence of SEQ ID NO:11, (ii) CDR-H2    comprising the amino acid sequence of SEQ ID NO:12, and (iii) CDR-H3    comprising the amino acid sequence of SEQ ID NO:13, and a a light    chain variable region (V_(L)FAP) comprising (iv) CDR-L1 comprising    the amino acid sequence of SEQ ID NO:14, (v) CDR-L2 comprising the    amino acid sequence of SEQ ID NO:15, and (vi) CDR-L3 comprising the    amino acid sequence of SEQ ID NO:16.

7. The bispecific antigen binding molecule of any one of paras 1 to 6,wherein the antigen binding domain capable of specific binding to FAPcomprises

-   (a) a heavy chain variable region (V_(H)FAP) comprising an amino    acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%    identical to the amino acid sequence of SEQ ID NO:9, and a light    chain variable region (V_(L)FAP) comprising an amino acid sequence    that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to    the amino acid sequence of SEQ ID NO:10, or-   (b) a heavy chain variable region (V_(H)FAP) comprising an amino    acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%    identical to the amino acid sequence of SEQ ID NO:17, and a light    chain variable region (V_(L)FAP) comprising an amino acid sequence    that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to    the amino acid sequence of SEQ ID NO:18.

8. The bispecific antigen binding molecule of any one of paras 1 to 7,wherein the antigen binding domain capable of specific binding to CD40comprises a heavy chain variable region (V_(H)CD40) comprising (i)CDR-H1 comprising the amino acid sequence of SEQ ID NO:19, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:20, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:21, and a light chainvariable region (V_(L)CD40) comprising (iv) CDR-L1 comprising the aminoacid sequence of SEQ ID NO:22, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO:23, and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO:24.

9. The bispecific antigen binding molecule of any one of paras 1 to 7,wherein the antigen binding domain capable of specific binding to CD40comprises a heavy chain variable region (V_(H)CD40) comprising (i)CDR-H1 comprising the amino acid sequence of SEQ ID NO:27, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:28, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:29, and a light chainvariable region (V_(L)CD40) comprising (iv) CDR-L1 comprising the aminoacid sequence of SEQ ID NO:30, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO:31, and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO:32.

10. The bispecific antigen binding molecule of any one of paras 1 to 9,wherein the antigen binding domain capable of specific binding to CD40comprises

(a) a VH comprising the amino acid sequence of SEQ ID NO:25 and a VLcomprising the amino acid sequence of SEQ ID NO:26, or

(b) a VH comprising the amino acid sequence of SEQ ID NO:33 and a VLcomprising the amino acid sequence of SEQ ID NO:34.

11. The bispecific antigen binding molecule of any one of paras 1 to 8,wherein the antigen binding domain capable of specific binding to CD40comprises

(i) a heavy chain variable region (V_(H)CD40) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55, and

(ii) a light chain variable region (V_(L)CD40) comprising the amino acidsequence selected from the group consisting of SEQ ID NO:56, SEQ IDNO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ IDNO:62, SEQ ID NO:63 and SEQ ID NO:64.

12. The bispecific antigen binding molecule of any one of paras 1 to 8or 11, wherein the antigen binding domain capable of specific binding toCD40 comprises a VH comprising the amino acid sequence of SEQ ID NO:47and a VL comprising the amino acid sequence of SEQ ID NO:57.

13. The bispecific antigen binding molecule of any one of paras 1 to 8,comprising

(i) at least one antigen binding domain capable of specific binding toCD40, comprising a heavy chain variable region (V_(H)CD40) comprising anamino acid sequence selected from the group consisting of SEQ ID NO:25,SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49,SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 andSEQ ID NO:55, and a light chain variable region (V_(L)CD40) comprisingan amino acid sequence selected from the group consisting of SEQ IDNO:26, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ IDNO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64, and

(ii) at least one antigen binding domain capable of specific binding toFAP, comprising a heavy chain variable region (V_(L)FAP) comprising anamino acid sequence of SEQ ID NO:9 and a light chain variable region(V_(L)FAP) comprising an amino acid sequence of SEQ ID NO:10, or a heavychain variable region (V_(L)FAP) comprising an amino acid sequence ofSEQ ID NO:17 and a light chain variable region (V_(L)FAP) comprising anamino acid sequence of SEQ ID NO:18.

14. The bispecific antigen binding molecule of any one of paras 2 to 13,wherein the Fc region is an IgG, particularly an IgG1 Fc region or anIgG4 Fc region.

15. The bispecific antigen binding molecule of any one of paras 2 to 14,wherein the Fc region comprises one or more amino acid substitution thatreduces the binding affinity of the antibody to an Fc receptor and/oreffector function.

16. The bispecific antigen binding molecule of any one of paras 2 to 15,wherein the Fc region is (i) of human IgG1 subclass with the amino acidmutations L234A, L235A and P329G (numbering according to Kabat EUindex), or (ii) of mouse IgG1 subclass with the amino acid mutationsD265A and P329G (numbering according to Kabat EU index).

17. The bispecific antigen binding molecule of any one of paras 2 to 16,wherein the Fc region comprises a modification promoting the associationof the first and second subunit of the Fc region.

18. The bispecific antigen binding molecule of any one of paras 2 to 17,wherein the first subunit of the Fc region comprises knobs and thesecond subunit of the Fc region comprises holes according to the knobsinto holes method.

19. The bispecific antibody of any one of paras 2 to 18, wherein

(i) the first subunit of the Fc region comprises the amino acidsubstitutions S354C and T366W (numbering according to Kabat EU index)and the second subunit of the Fc region comprises the amino acidsubstitutions Y349C, T366S and Y407V (numbering according to Kabat EUindex), or

(ii) the first subunit of the Fc region comprises the amino acidsubstitutions K392D and K409D (numbering according to Kabat EU index)and the second subunit of the Fc region comprises the amino acidsubstitutions E356K and:D⁻399K (numbering according to Kabat EU index).

20. The bispecific antigen binding molecule of any one of paras 1 to 19,wherein the bispecific antigen binding molecule comprises

(a) at least two Fab fragments capable of specific binding to CD40connected to a Fc region, and

(b) at least one antigen binding domain capable of specific binding toFAP connected to the C-terminus of the Fc region.

21. The bispecific antigen binding molecule of any one of paras 1 to 19,wherein the bispecific antigen binding molecule comprises

(a) two light chains and two heavy chains of an antibody comprising twoFab fragments capable of specific binding to CD40, and a Fc region, and

(b) a VH and a VL of an antigen binding domain capable specific bindingto FAP, wherein the VH is connected to the C-terminus of one of the twoheavy chains of (a), and wherein the VL is connected to the C-terminusof the other of the two heavy chains of (a).

22. The bispecific antigen binding molecule of any one of paras 1 to 19,wherein the bispecific antigen binding molecule comprises

(a) two light chains and two heavy chains of an antibody comprising twoFab fragments capable of specific binding to CD40, and a Fc region, and

(b) two Fab fragments capable of specific binding to FAP, wherein one ofthe Fab fragments is connected to the C-terminus of one of the two heavychains of (a), and the other of the Fab fragments is connected to theC-terminus of the other of the two heavy chains of (a).

23. The bispecific antigen binding molecule of any one of paras 1 to 19,wherein the bispecific antigen binding molecule comprises

(a) two heavy chains, each heavy chain comprising a VH and CH1 domain ofa Fab fragment capable of specific binding to CD40 and a Fc regionsubunit,

(b) two light chains, each light chain comprising a VL and CL domain ofa Fab fragment capable of specific binding to CD40, and

(c) a VH and a VL of an antigen binding domain capable of specificbinding to FAP, wherein the VH is connected to the C-terminus of one ofthe two heavy chains of (a), and wherein the VL is connected to theC-terminus of the other of the two heavy chains of (a).

24. The bispecific antigen binding molecule of any one of paras 1 to 19,wherein the bispecific antigen binding molecule comprises

(a) two heavy chains, each heavy chain comprising a VH and CH1 domain ofa Fab fragment capable of specific binding to CD40, and a Fc regionsubunit,

(b) two light chains, each light chain comprising a VL and CL domain ofa Fab fragment capable of specific binding to CD40, and

(c) two Fab fragments capable of specific binding to FAP, wherein one ofthe Fab fragments is connected to the C-terminus of one of the two heavychains of (a), and the other of the Fab fragments is connected to theC-terminus of the other of the two heavy chains of (a).

25. The bispecific antigen binding molecule of any one of paras 1 to 19,wherein the bispecific antigen binding molecule comprises

(a) two heavy chains, each heavy chain comprising a VH and CH1 domain ofa Fab fragment capable of specific binding to CD40, and a Fc regionsubunit,

(b) two light chains, each light chain comprising a VL and CL domain ofa Fab fragment capable of specific binding to CD40, and

(c) one Fab fragment capable of specific binding to FAP, wherein the Fabfragments is connected to the C-terminus of one of the two heavy chainsof (a).

26. The bispecific antigen binding molecule of any one of paras 22 to25, wherein the Fab fragment or the two Fab fragments capable ofspecific binding to FAP are crossover Fab fragments each comprising aVL-CH1 chain and a VH-CL chain, and wherein the VL-CH1 chain isconnected to the C-terminus of one of the two heavy chains of (a).

27. The bispecific antigen binding molecule of any one of paras 1 to 26,wherein the bispecific antigen binding molecule comprises four Fabfragments capable of specific binding to CD40.

28. The bispecific antigen binding molecule of any one of paras 23 to27, wherein each of the two heavy chains of (a) comprises two VHs andtwo CH1 domains of a Fab fragment capable of specific binding to CD40.

29. The bispecific antigen binding molecule of any one of paras 23 to28, wherein one or more of the Fab fragments capable of specific bindingto CD40 comprises

a CL domain comprising an arginine (R) at amino acid at position 123(numbering according to Kabat EU index) and a lysine (K) at amino acidat position 124 (numbering according to Kabat EU index), and

a CH1 domain comprising a glutamic acid (E) at amino acid at position147 (numbering according to Kabat EU index) and a glutamic acid (E) atamino acid at position 213 (numbering according to Kabat EU index).

30. A polynucleotide encoding the bispecific antigen binding molecule ofany one of paras 1 to 29.

31. An expression vector comprising the polynucleotide of claim 30.

32. A host cell comprising the polynucleotide of para 30 or theexpression vector of para 31.

33. A method of producing a bispecific antigen binding molecule,comprising culturing the host cell of para 32 under conditions suitablefor the expression of the bispecific antigen binding molecule, andisolating the bispecific antigen binding molecule.

34. A pharmaceutical composition comprising the bispecific antigenbinding molecule of any one of paras 1 to 29 and at least onepharmaceutically acceptable excipient.

35. The bispecific antigen binding molecule of any one of paras 1 to 29,or the pharmaceutical composition of para 34, for use as a medicament.

36. The bispecific antigen binding molecule of any one of paras 1 to 29,or the pharmaceutical composition of para 34, for use

-   (i) in inducing immune stimulation by CD40⁺ antigen-presenting cells    (APCs),-   (ii) in stimulating tumor-specific T cell response,-   (iii) in causing apoptosis of tumor cells,-   (iv) in the treatment of cancer,-   (v) in delaying progression of cancer,-   (vi) in prolonging the survival of a patient suffering from cancer,-   (vii) in the treatment of infections.

37. The bispecific antigen binding molecule of any one of paras 1 to 29,or the pharmaceutical composition of para 34, for use in the treatmentof cancer.

38. Use of the bispecific antigen binding molecule of any one of paras 1to 29, or the pharmaceutical composition of para 34, in the manufactureof a medicament for the treatment of cancer.

39. A method of treating an individual having cancer comprisingadministering to the individual an effective amount of the bispecificantigen binding molecule of any one of paras 1 to 29, or thepharmaceutical composition of para 34.

40. The bispecific antigen binding molecule of any one of paras 1 to 29,or the pharmaceutical composition of para 34, for use in up-regulatingor prolonging cytotoxic T cell activity.

41. The bispecific antigen binding molecule according to any one ofparas 1 to 29 or the pharmaceutical composition according to para 34 foruse in the treatment of cancer, wherein the bispecific antigen bindingmolecule is administered in combination with a chemotherapeutic agent,radiation and/or other agents for use in cancer immunotherapy.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook etal., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions. General information regarding the nucleotide sequences ofhuman immunoglobulin light and heavy chains is given in: Kabat, E. A. etal., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed.,NIH Publication No 91-3242.

DNA Sequencing

DNA sequences were determined by double strand sequencing.

Gene Synthesis

Desired gene segments were either generated by PCR using appropriatetemplates or were synthesized by Geneart AG (Regensburg, Germany) fromsynthetic oligonucleotides and PCR products by automated gene synthesis.In cases where no exact gene sequence was available, oligonucleotideprimers were designed based on sequences from closest homologues and thegenes were isolated by RT-PCR from RNA originating from the appropriatetissue. The gene segments flanked by singular restriction endonucleasecleavage sites were cloned into standard cloning/sequencing vectors. Theplasmid DNA was purified from transformed bacteria and concentrationdetermined by UV spectroscopy. The DNA sequence of the subcloned genefragments was confirmed by DNA sequencing. Gene segments were designedwith suitable restriction sites to allow sub-cloning into the respectiveexpression vectors. All constructs were designed with a 5′-end DNAsequence coding for a leader peptide which targets proteins forsecretion in eukaryotic cells.

Protein Purification

Proteins were purified from filtered cell culture supernatants referringto standard protocols. In brief, antibodies were applied to a Protein ASepharose column (GE healthcare) and washed with PBS. Elution ofantibodies was achieved at pH 2.8 followed by immediate neutralizationof the sample. Aggregated protein was separated from monomericantibodies by size exclusion chromatography (Superdex 200, GEHealthcare) in PBS or in 20 mM Histidine, 150 mM NaCl pH 6.0. Monomericantibody fractions were pooled, concentrated (if required) using e.g., aMILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen andstored at −20° C. or −80° C. Part of the samples were provided forsubsequent protein analytics and analytical characterization e.g. bySDS-PAGE, size exclusion chromatography (SEC) or mass spectrometry.

SDS-PAGE

The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to themanufacturer's instruction. In particular, 10% or 4-12% NuPAGE® Novex®Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, withNuPAGE® Antioxidant running buffer additive) or MOPS (non-reduced gels)running buffer was used.

CE-SDS

Purity, antibody integrity and molecular weight of bispecific andcontrol antibodies were analyzed by CE-SDS using microfluidic Labchiptechnology (Caliper Life Science, USA). 5 μl of protein solution wasprepared for CE-SDS analysis using the HT Protein Express Reagent Kitaccording manufacturer's instructions and analysed on LabChip GXIIsystem using a HT Protein Express Chip. Data were analyzed using LabChipGX Software version 3.0.618.0.

Analytical Size Exclusion Chromatography

Size exclusion chromatography (SEC) for the determination of theaggregation and oligomeric state of antibodies was performed by HPLCchromatography. Briefly, Protein A purified antibodies were applied to aTosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH₂PO₄/K₂HPO₄, pH 7.5on an Agilent HPLC 1100 system or to a Superdex 200 column (GEHealthcare) in 2× PBS on a Dionex HPLC-System. The eluted protein wasquantified by UV absorbance and integration of peak areas. BioRad GelFiltration Standard 151-1901 served as a standard.

Mass Spectrometry

This section describes the characterization of the multispecificantibodies with VH/VL or CH/CL exchange (CrossMabs) with emphasis ontheir correct assembly. The expected primary structures were analyzed byelectrospray ionization mass spectrometry (ESI-MS) of the deglycosylatedintact CrossMabs and deglycosylated/FabALACTICA or alternativelydeglycosylated/GingisKHAN digested CrossMabs.

The CrossMabs were deglycosylated with N-Glycosidase F in a phosphate orTris buffer at 37° C. for up to 17 h at a protein concentration of 1mg/ml. The FabALACTICA or GingisKHAN (Genovis AB; Sweden) digestionswere performed in the buffers supplied by the vendor with 100 μgdeglycosylated CrossMabs. Prior to mass spectrometry the samples weredesalted via HPLC on a Sephadex G25 column (GE Healthcare). The totalmass was determined via ESI-MS on a maXis 4G UHR-QTOF MS system (BrukerDaltonik) equipped with a TriVersa NanoMate source (Advion).

Example 1 Generation and Production of Bispecific Constructs TargetingCD40 and Fibroblast Activation Protein (FAP) 1.1 Generation ofBispecific Antigen Binding Molecules Targeting CD40 and FibroblastActivation Protein (FAP)

The cDNAs encoding variable heavy and light chain domains of the antiCD40 binder (SEQ ID NO:10 and SEQ ID NO:16 of WO 2006/128103) werecloned in frame with the corresponding constant heavy or light chains ofhuman IgG1 in suitable expression plasmids. Expression of heavy andlight chain is driven by a chimeric MPSV promoter consisting of the MPSVcore promoter and a CMV enhancer element. The expression cassette alsocontains a synthetic polyA signal at the 3′ end of the cDNAs. Inaddition the plasmid vectors harbor an origin of replication (EBV OriP)for episomal maintenance of the plasmids. Amino acid and nucleotidesequences of the variable domains of the CD40 mAb and the FAP mAb areshown in Table 1 and 2, respectively.

Different bispecific CD40-FAP antibodies have been prepared in 4+1 and4+2 formats consisting of four CD40 binding moieties combined witheither one or two FAP binding arms at the C-terminus of an Fc or in 2+1and 2+2 formats consisting of two CD40 binding moieties combined witheither one or two FAP binding arms at the C-terminus of an Fc (FIG. 1A,FIG. 1B, FIG. 1C, and FIG. 1D). The generation and preparation of FAPbinder 28H1 is described in WO 2012/020006 A2, which is incorporatedherein by reference. To generate the 4+1 and the 2+1 molecules theknob-into-hole technology was used to achieve heterodimerization. TheS354C/T366W mutations were introduced in the first heavy chain HC1 (Fcknob heavy chain) and the Y349C/T366S/L368A/Y407V mutations wereintroduced in the second heavy chain HC2 (Fc hole heavy chain). In the4+2 and 2+2 molecules the CrossMab technology as described in WO2010/145792 Al ensured correct light chain pairing. Independent of thebispecific format, in all cases an effector silent Fc (P329G; L234,L235A) was used to abrogate binding to Fcγ receptors according to themethod described in WO 2012/130831 A1. Sequences of the bispecificmolecules are shown in Table 3 and 4.

Besides molecules targeting the human receptors also surrogate moleculeswere generated in the same formats that recognize the murine antigens.In these cases the heterodimerization of 4+1 molecules was achieved byDD/KK mutations (introduction of the Lys392Asp and Lys409Asp in thefirst heavy chain and introduction of the Glu356Lys and Asp399Lysmutation in the second heavy chain) in the Fc according to the methoddescribed in by Gunasekaran et al., J. Biol. Chem. 2010,19637-19646,while binding to Fc receptors was inhibited by D270A/P329G mutations inaccordance with the method described in Baudino et al., J. Immunol.(2008), 181, 6664-9. or in WO 2016/030350 A1.

All genes were transiently expressed under control of a chimeric MPSVpromoter consisting of the MPSV core promoter combined with the CMVpromoter enhancer fragment. The expression vector also contains the oriPregion for episomal replication in EBNA (Epstein Barr Virus NuclearAntigen) containing host cells.

TABLE 1 Amino acid sequences of the variable domains of the CD40antibodies, the FAP antibody and DP47 antibody Seq ID DescriptionSequence No hu CD40 VH EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 25GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL RAEDTAVYYCAREGIYWWGQGTLVTVSShu CD40 VL DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 26KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE DFATYFCSQTTHVPWTFGQGTKVEIKFAP 28H1 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 9GLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSS FAP 28H1 VLEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQ 10APRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV YYCQQGQVIPPTFGQGTKVEIK DP47VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK 71GLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSS DP47 VLEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQ 72APRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV YYCQQYGSSPLTFGQGTKVEIK muCD40 VH EVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTK 33GLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCGRHSSYFDYWGQGVMVTVSS mu CD40 VLDTVLTQSPALAVSPGERVTISCRASDSVSTLMHWYQQKPGQQP 34KLLIYLASHLESGVPARFSGSGSGTDFTLTIDPVEADDTATYY CQQSWNDPWTFGGGTKLELK

TABLE 2 Nucleotide sequences of the variable domains of the CD40antibodies, the FAP antibody and DP47 antibody Seq ID DescriptionSequence No hu CD40 VH GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 73GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTTCACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAACCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGTGGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCATCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGC hu CD40 VLGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCG 74TGGGCGACAGAGTGACCATCACCTGTCGGAGCAGCCAGAGCCTGGTGCACAGCAACGGCAACACCTTCCTGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACACCGTGTCCAACCGGTTCAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTTCACCCTGACAATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTATTTCTGCAGCCAGACCACCCACGTGCCCTGGACATTTGGACAGGGCACCAAGGTGGAAATCAAG FAP 28H1 VHGAGGTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGCAGCCTG 75GCGGATCTCTGAGACTGTCCTGCGCCGCCTCCGGCTTCACCTTCTCCTCCCACGCCATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGACTCTGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGGCTGGCTGGGCAACTTCGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTC CAGC FAP 28H1 VLGAGATCGTGCTGACCCAGTCTCCCGGCACCCTGAGCCTGAGCC 76CTGGCGAGAGAGCCACCCTGAGCTGCAGAGCCAGCCAGAGCGTGAGCCGGAGCTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCATCGGCGCCAGCACCCGGGCCACCGGCATCCCCGATAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCCAGGTGATCCCCCCCACCTTCGGCC AGGGCACCAAGGTGGAAATCAAG DP47VH GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTG 77GGGGGTCCCTGAGACTCTCCTGTGCAGCCAGCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGCAGCGGATTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAG C DP47 VLGAAATTGTGCTGACCCAGAGCCCCGGCACCCTGTCACTGTCTC 78CAGGCGAAAGAGCCACCCTGAGCTGCAGAGCCAGCCAGAGCGTGTCCAGCTCTTACCTGGCCTGGTATCAGCAGAAGCCCGGACAGGCCCCCAGACTGCTGATCTACGGCGCCTCTTCTAGAGCCACCGGCATCCCCGATAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACAATCAGCAGACTGGAACCCGAGGACTTTGCCGTGTATTACTGCCAGCAGTACGGCAGCAGCCCCCTGACCTTTGGCC AGGGCACCAAGGTGGAAATCAAA muCD40 VH GAAGTGCAGCTGGTGGAATCCGACGGCGGACTGGTGCAGCCTG 79GCAGATCTCTGAAGCTGCCTTGTGCCGCCTCCGGCTTCACCTTCTCCGACTACTACATGGCCTGGGTGCGACAGGCCCCTACCAAGGGACTGGAATGGGTGGCCTCCATCTCCTACGACGGCTCCTCCACCTACTACCGGGACTCTGTGAAGGGCCGGTTCACCATCTCTCGGGACAACGCCAAGTCCACCCTGTACCTGCAGATGGACTCCCTGCGGAGCGAGGACACCGCTACCTACTACTGCGGCAGACACTCCTCCTACTTCGACTACTGGGGCCAGGGCGTGATGGTCACCGTGTC CTCT mu CD40 VLGACACTGTACTGACCCAGTCTCCTGCTTTGGCTGTGTCTCCAG 80GAGAGAGGGTTACCATCTCCTGTAGGGCCAGTGACAGTGTCAGTACACTTATGCACTGGTACCAACAGAAACCAGGACAGCAACCCAAACTCCTCATCTATCTAGCATCACACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATTGATCCTGTGGAGGCTGATGACACTGCAACCTATTACTGTCAGCAGAGTTGGAATGATCCGTGGACGTTCGGTGGAGGCA CCAAGCTGGAATTGAAA

TABLE 3 Amino acid sequences of the CD40 IgG and the bispecific antigenbinding molecules Seq ID Construct Sequence No P1AD4470 CD40 IgG CD40IgG EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 81 Heavy chainGLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG CD40 lightDIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 82 chainKPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AD4453 CD40 x FAP(28H1) (4 + 1) CD40 VHCH1- EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK83 CD40 VHCH1- GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSLFcknob_PGLALA- RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS 28H1 VHKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGKGLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSS CD40 VHCH1-EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 84 CD40 VHCH1-GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL Fchole_PGLALA-RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS 28H1 VLKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGKGLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPED FAVYYCQQGQVIPPTFGQGTKVEIKCD40 light DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 82 chainKPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AD4455 CD40 x FAP(28H1) (4 + 2) CD40 VHCH1- EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK85 CD40 VHCH1- GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL Fc_PGLALA-RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS 28H1 VLCH1KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ ‘EE’SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGKGLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS C CD40-lightDIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 86 chain;KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE ,RK’DFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 28H1 VHCLEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 87GLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC P1AA9641 CD40 x FAP(28H1) (2 + 1) CD40_Fc EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 88hole_PGLALA_28H1 GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL VLRAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIP PTFGQGTKVEIKhuCD40_Fcknob_PGLALA_28H1 EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 89VH GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA KGWLGNFDYWGQGTLVTVSS + CD40LC DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 82KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AA9663 CD40 x FAP(28H1) (2 + 2) CD40-28H1 EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 902 + 2; ,EE’ GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL huCD40-RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS Fc_PGLALA_28H1_VLCH1KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ ‘EE’SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC + CD40DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 86 LC; ,RK’KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC + 28H1 VHCLEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 87GLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC P1AD4574 CD40 xDP47 (4 + 1) CD40 VHCH1- EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 91CD40 VHCH1- GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL Fcknob_PGLALA-RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS DP47 VHKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGKGLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSS CD40 VHCH1-EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 92 CD40 VHCH1-GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL Fchole_PGLALA-RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS DP47 VLKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGKGLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPED FAVYYCQQYGSSPLTFGQGTKVEIK+ CD40 light DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 82 chainKPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AD4465 CD40 x DP47 (4+ 2) CD40 VHCH1- EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 93 CD40VHCH1- GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL Fc_PGLALA-RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS DP47 VLCH1KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ ‘EE’SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGKGLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS C + CD40 lightDIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 86 chain; ‘RK’KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC + DP47VHCLEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK 94GLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC P1AD4520 or P1AD9139mu CD40 (FGK4.5) x FAP (28H1) (4 + 1) muCD40EVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTK 95 VHCH1-GLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSL muCD40RSEDTATYYCGRHSSYFDYWGQGVMVTVSSAKTTPPSVYPLAP VHCH1-GSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAV FcKK_DAPG-LQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVP 28H1 VHRDCGGGGSGGGGSEVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTKGLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCGRHSSYFDYWGQGVMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPIEKTISKTKGRPKAPQVYTIPPPKKQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMKTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSS muCD40EVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTK 96 VHCH1-GLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSL muCD40RSEDTATYYCGRHSSYFDYWGQGVMVTVSSAKTTPPSVYPLAP VHCH1-GSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAV FcDD_DAPG-LQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVP 28H1 VLRDCGGGGSGGGGSEVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTKGLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCGRHSSYFDYWGQGVMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYDNTQPIMDTDGSYFVYSDLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVY YCQQGQVIPPTFGQGTKVEIK muCD40 light DTVLTQSPALAVSPGERVTISCRASDSVSTLMHWYQQKPGQQP 97 chainKLLIYLASHLESGVPARFSGSGSGTDFTLTIDPVEADDTATYYCQQSWNDPWTFGGGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC P1AD4558 mu CD40 (FGK5.4) xFAP (28H1) (4 + 2) muCD40 EVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTK 98VHCH1- GLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSL muCD40RSEDTATYYCGRHSSYFDYWGQGVMVTVSSAKTTPPSVYPLAP VHCH1-GSAAQTNSMVTLGCLVEGYFPEPVTVTWNSGSLSSGVHTFPAV Fc_DAPG-28H1LQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDEKIVP VLCH1 ‘EE’RDCGGGGSGGGGSEVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTKGLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCGRHSSYFDYWGQGVMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVEGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDEKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDC 28H1 VHCLEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 99 (mu)GLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIV KSFNRNEC mu CD40 lightDTVLTQSPALAVSPGERVTISCRASDSVSTLMHWYQQKPGQQP 100 chain; ‘RK’KLLIYLASHLESGVPARFSGSGSGTDFTLTIDPVEADDTATYYCQQSWNDPWTFGGGTKLELKRADAAPTVSIFPPSSRKLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC P1AD4521 mu CD40-DP47 (4 + 1)muCD40 EVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTK 101 VHCH1-GLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSL muCD40RSEDTATYYCGRHSSYFDYWGQGVMVTVSSAKTTPPSVYPLAP VHCH1-GSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAV FcKK_DAPG-LQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVP DP47 VHRDCGGGGSGGGGSEVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTKGLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCGRHSSYFDYWGQGVMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPIEKTISKTKGRPKAPQVYTIPPPKKQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMKTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCAKGSGFDYWGQGTLVTVSSmuCD40 EVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTK 102 VHCH1-muGLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSL CD40 VHCH1-RSEDTATYYCGRHSSYFDYWGQGVMVTVSSAKTTPPSVYPLAP FcDD_DAPG-GSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAV DP47 VLLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGGGGSGGGGSEVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTKGLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCGRHSSYFDYWGQGVMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYDNTQPIMDTDGSYFVYSDLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVY YCQQYGSSPLTFGQGTKVEIK + muCD40 DTVLTQSPALAVSPGERVTISCRASDSVSTLMHWYQQKPGQQP 97 light chainKLLIYLASHLESGVPARFSGSGSGTDFTLTIDPVEADDTATYYCQQSWNDPWTFGGGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC P1AD4555 mu CD40-DP47 (4 + 2)muCD40 EVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTK 103 VHCH1-GLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSL muCD40RSEDTATYYCGRHSSYFDYWGQGVMVTVSSAKTTPPSVYPLAP VHCH1-GSAAQTNSMVTLGCLVEGYFPEPVTVTWNSGSLSSGVHTFPAV Fc_DAPG-LQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDEKIVP DP47 VLCH1RDCGGGGSGGGGSEVQLVESDGGLVQPGRSLKLPCAASGFTFS ‘EE’DYYMAWVRQAPTKGLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCGRHSSYFDYWGQGVMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVEGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDEKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIKSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDC + mu CD40DTVLTQSPALAVSPGERVTISCRASDSVSTLMHWYQQKPGQQP 100 light chain;KLLIYLASHLESGVPARFSGSGSGTDFTLTIDPVEADDTATYY ,RK’CQQSWNDPWTFGGGTKLELKRADAAPTVSIFPPSSRKLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC DP47 VHCLEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK 104 (mu)GLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSASDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVK SFNRNEC

TABLE 4 Nucleotide sequences of the bispecific antigen binding moleculesSeq ID Construct Sequence No CD40 IgGGAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 105 Heavy chainGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTTCACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAACCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGTGGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCATCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGCGCTAGCACCAAGGGCCCAAGCGTGTTCCCACTGGCCCCAAGCAGCAAGTCTACCAGCGGAGGAACAGCCGCCCTGGGATGTCTGGTGAAGGACTACTTCCCCGAGCCAGTGACAGTGAGCTGGAACTCTGGCGCCCTGACATCTGGCGTGCACACATTCCCAGCCGTGCTGCAGTCTAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCAAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCACCATGTCCAGCCCCAGAGCTGCTGGGAGGACCTAGCGTGTTCCTGTTCCCCCCCAAGCCAAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACATGTGTGGTGGTGGACGTGTCTCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGAGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACCGCGTGGTGTCTGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCAATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCAAGGGAGCCACAGGTGTACACCCTGCCCCCATCTAGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA CD40 lightGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCG 106 chainTGGGCGACAGAGTGACCATCACCTGTCGGAGCAGCCAGAGCCTGGTGCACAGCAACGGCAACACCTTCCTGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACACCGTGTCCAACCGGTTCAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTTCACCCTGACAATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTATTTCTGCAGCCAGACCACCCACGTGCCCTGGACATTTGGACAGGGCACCAAGGTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAAC AGGGGAGAGTGT CD40 x FAP(28H1) (4 + 1) CD40 GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 107VHCH1-CD40 GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTT VHCH1-CACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAG Fchole_PGLALA-GGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAA 28H1 VHCCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGTGGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCATCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGCGCTTCCACCAAGGGCCCTAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGTCTACCAGCGGAGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACTGTGCCCAGCAGCAGCCTGGGAACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACGGCGGAGGCGGATCAGGCGGCGGAGGATCCGAAGTGCAGCTGGTGGAAAGTGGGGGAGGCCTGGTGCAGCCAGGGGGAAGCCTGAGACTGTCTTGTGCCGCTTCCGGCTACTCTTTTACCGGGTATTATATCCATTGGGTGCGGCAGGCTCCAGGGAAAGGCCTGGAATGGGTGGCACGCGTGATCCCTAACGCAGGCGGCACCTCTTATAATCAGAAGTTTAAAGGGCGCTTTACCCTGTCCGTGGACAATTCCAAGAATACTGCTTACCTGCAGATGAATTCCCTGCGCGCCGAAGATACAGCTGTGTATTACTGCGCCAGAGAAGGGATCTATTGGTGGGGACAGGGCACCCTCGTGACAGTGTCATCCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCCGGAGGAGGGGGAAGTGGCGGCGGAGGATCTGAGGTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGCAGCCTGGCGGATCTCTGAGACTGTCCTGCGCCGCCTCCGGCTTCACCTTCTCCTCCCACGCCATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGACTCTGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGGCTGGCTGGGCAACTTCGACTACTGGGGCCAGGGCACCCTGGT CACCGTGTCCAGC CD40GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 108 VHCH1-CD40GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTT VHCH1-CACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAG Fchole_PGLALA-GGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAA 28H1 VLCCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGTGGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCATCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGCGCTTCCACCAAGGGCCCTAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGTCTACCAGCGGAGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACTGTGCCCAGCAGCAGCCTGGGAACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACGGCGGAGGCGGATCAGGCGGCGGAGGATCCGAAGTGCAGCTGGTGGAAAGTGGGGGAGGCCTGGTGCAGCCAGGGGGAAGCCTGAGACTGTCTTGTGCCGCTTCCGGCTACTCTTTTACCGGGTATTATATCCATTGGGTGCGGCAGGCTCCAGGGAAAGGCCTGGAATGGGTGGCACGCGTGATCCCTAACGCAGGCGGCACCTCTTATAATCAGAAGTTTAAAGGGCGCTTTACCCTGTCCGTGGACAATTCCAAGAATACTGCTTACCTGCAGATGAATTCCCTGCGCGCCGAAGATACAGCTGTGTATTACTGCGCCAGAGAAGGGATCTATTGGTGGGGACAGGGCACCCTCGTGACAGTGTCATCCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAAGCGGAGGAGGAGGATCCGGTGGTGGCGGATCTGGGGGCGGTGGATCTGAGATCGTGCTGACCCAGTCTCCCGGCACCCTGAGCCTGAGCCCTGGCGAGAGAGCCACCCTGAGCTGCAGAGCCAGCCAGAGCGTGAGCCGGAGCTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCATCGGCGCCAGCACCCGGGCCACCGGCATCCCCGATAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCCAGGTGATCCCCCCCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG CD40 light see above 106 chain CD40 xFAP (28H1) (4 + 2) CD40 GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 109VHCH1-CD40 GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTT VHCH1-CACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAG Fc_PGLALA-GGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAA 28H1 VLCH1CCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGT ‘EE’GGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCATCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGCGCTTCTACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGGAGGACTACTTTCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTCCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACTGTGCCCAGCAGCAGCCTGGGAACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAACCCAAGAGCTGCGACGGCGGAGGCGGATCTGGCGGCGGAGGATCCGAAGTGCAGCTGGTGGAAAGTGGGGGAGGCCTGGTGCAGCCAGGGGGAAGCCTGAGACTGTCTTGTGCCGCTTCCGGCTACTCTTTTACCGGGTATTATATCCATTGGGTGCGGCAGGCTCCAGGGAAAGGCCTGGAATGGGTGGCACGCGTGATCCCTAACGCAGGCGGCACCTCTTATAATCAGAAGTTTAAAGGGCGCTTTACCCTGTCCGTGGACAATTCCAAGAATACTGCTTACCTGCAGATGAATTCCCTGCGCGCCGAAGATACAGCTGTGTATTACTGCGCCAGAGAAGGGATCTATTGGTGGGGACAGGGCACCCTCGTGACAGTGTCATCCGCTAGCACCAAGGGACCTTCCGTGTTTCCCCTGGCTCCCAGCTCCAAGTCTACCTCTGGGGGCACAGCTGCTCTGGGATGTCTGGTGGAAGATTATTTTCCTGAACCTGTGACCGTGTCATGGAACAGCGGAGCCCTGACCTCCGGGGTGCACACATTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACACAGACATATATCTGTAATGTGAATCACAAACCCTCTAATACCAAAGTGGATGAGAAAGTGGAACCTAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCATCTGTGTTTCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCAGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCGCGGGAAGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGAGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCTCAGGTGTACACCCTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACACAGAAGTCTCTGAGCCTGAGCCCTGGCGGAGGGGGAGGATCTGGGGGAGGCGGAAGTGGGGGAGGGGGTTCCGGAGGCGGCGGATCAGAAATTGTGCTGACCCAGTCCCCCGGCACCCTGTCACTGTCTCCAGGCGAAAGAGCCACCCTGAGCTGTAGGGCCTCCCAGAGCGTGTCCAGAAGCTATCTGGCCTGGTATCAGCAGAAGCCCGGACAGGCCCCCAGACTGCTGATCATTGGCGCCTCTACCAGAGCCACCGGCATCCCCGATAGATTCAGCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCTCCAGACTGGAACCCGAGGACTTTGCCGTGTACTATTGCCAGCAGGGCCAAGTGATCCCCCCCACCTTTGGCCAGGGAACAAAGGTGGAAATCAAGTCCAGCGCTTCCACCAAGGGCCCCTCAGTGTTCCCACTGGCACCATCCAGCAAGTCCACAAGCGGAGGAACCGCCGCTCTGGGCTGTCTCGTGAAAGACTACTTTCCAGAGCCAGTGACCGTGTCCTGGAATAGTGGCGCTCTGACTTCTGGCGTGCACACTTTCCCCGCAGTGCTGCAGAGTTCTGGCCTGTACTCCCTGAGTAGCGTCGTGACAGTGCCCTCCTCTAGCCTGGGCACTCAGACTTACATCTGCAATGTGAATCATAAGCCTTCCAACACAAAAGTGGACAAAAAAGTGGAACCCAAATCT TGC CD40-lightGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCG 110 chain;TGGGCGACAGAGTGACCATCACCTGTCGGAGCAGCCAGAGCCT ,RK’GGTGCACAGCAACGGCAACACCTTCCTGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACACCGTGTCCAACCGGTTCAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTTCACCCTGACAATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTATTTCTGCAGCCAGACCACCCACGTGCCCTGGACATTTGGACAGGGCACCAAGGTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATCGGAAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAAC AGGGGAGAGTGT 28H1 VHCLGAAGTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGCAGCCTG 111GCGGATCTCTGAGACTGTCCTGCGCCGCCTCCGGCTTCACCTTCTCCTCCCACGCCATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGACTCTGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGGCTGGCTGGGCAACTTCGACTACTGGGGACAGGGCACCCTGGTCACCGTGTCCAGCGCTAGCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACAGCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACC AAGTCTTTCAACCGGGGCGAGTGCCD40 x FAP (28H1) (2 + 1) pETR17111GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 112huCD40_Fchole_PGLALA_28H1 GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTT VLCACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAACCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGTGGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCATCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAAGCGGAGGAGGAGGATCCGGTGGTGGCGGATCTGGGGGCGGTGGATCTGAGATCGTGCTGACCCAGTCTCCCGGCACCCTGAGCCTGAGCCCTGGCGAGAGAGCCACCCTGAGCTGCAGAGCCAGCCAGAGCGTGAGCCGGAGCTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCATCGGCGCCAGCACCCGGGCCACCGGCATCCCCGATAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCCAGGTGATCCCCCCCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAGTGA pETR17112GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 113huCD40_Fcknob_PGLALA_28H1 GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTT VHCACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAACCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGTGGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCATCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCCGGAGGAGGGGGAAGTGGCGGCGGAGGATCTGAGGTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGCAGCCTGGCGGATCTCTGAGACTGTCCTGCGCCGCCTCCGGCTTCACCTTCTCCTCCCACGCCATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGACTCTGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGGCTGGCTGGGCAACTTCGACTACTGGGGCCAGGGCACCC TGGTCACCGTGTCCAGCTGA +CD40LC see above 106 pETR15390 CD40 x FAP (28H1) (2 + 2) pETR17113GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 114 huCD40-GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTT Fc_PGLALA_28H1_VLCH1CACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAG ‘EE’GGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAACCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGTGGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCATCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGCGCTAGCACCAAGGGACCTTCCGTGTTTCCCCTGGCTCCCAGCTCCAAGTCTACCTCTGGGGGCACAGCTGCTCTGGGATGTCTGGTGGAAGATTATTTTCCTGAACCTGTGACCGTGTCATGGAACAGCGGAGCCCTGACCTCCGGGGTGCACACATTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACACAGACATATATCTGTAATGTGAATCACAAACCCTCTAATACCAAAGTGGATGAGAAAGTGGAACCTAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCATCTGTGTTTCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCAGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCGCGGGAAGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGAGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCTCAGGTGTACACCCTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACACAGAAGTCTCTGAGCCTGAGCCCTGGCGGAGGGGGAGGATCTGGGGGAGGCGGAAGTGGGGGAGGGGGTTCCGGAGGCGGCGGATCAGAAATTGTGCTGACCCAGTCCCCCGGCACCCTGTCACTGTCTCCAGGCGAAAGAGCCACCCTGAGCTGTAGGGCCTCCCAGAGCGTGTCCAGAAGCTATCTGGCCTGGTATCAGCAGAAGCCCGGACAGGCCCCCAGACTGCTGATCATTGGCGCCTCTACCAGAGCCACCGGCATCCCCGATAGATTCAGCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCTCCAGACTGGAACCCGAGGACTTTGCCGTGTACTATTGCCAGCAGGGCCAAGTGATCCCCCCCACCTTTGGCCAGGGAACAAAGGTGGAAATCAAGTCCAGCGCTTCCACCAAGGGCCCCTCAGTGTTCCCACTGGCACCATCCAGCAAGTCCACAAGCGGAGGAACCGCCGCTCTGGGCTGTCTCGTGAAAGACTACTTTCCAGAGCCAGTGACCGTGTCCTGGAATAGTGGCGCTCTGACTTCTGGCGTGCACACTTTCCCCGCAGTGCTGCAGAGTTCTGGCCTGTACTCCCTGAGTAGCGTCGTGACAGTGCCCTCCTCTAGCCTGGGCACTCAGACTTACATCTGCAATGTGAATCATAAGCCTTCCAACACAAAAGTGGACAAAAAAGTGGAACCCAA ATCTTGCTGA +CD40 see above110 LC; ,RK’ pETR15391 +28H1 VHCL see above 111 pETR15114 CD40 x DP47 (4+ 1) CD40 GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 115 VHCH1-CD40GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTT VHCH1-CACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAG Fcknob_PGLALA-GGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAA DP47 VHCCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGTGGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCATCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGCGCTTCCACCAAGGGCCCTAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGTCTACCAGCGGAGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACTGTGCCCAGCAGCAGCCTGGGAACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACGGCGGAGGCGGATCAGGCGGCGGAGGATCCGAAGTGCAGCTGGTGGAAAGTGGGGGAGGCCTGGTGCAGCCAGGGGGAAGCCTGAGACTGTCTTGTGCCGCTTCCGGCTACTCTTTTACCGGGTATTATATCCATTGGGTGCGGCAGGCTCCAGGGAAAGGCCTGGAATGGGTGGCACGCGTGATCCCTAACGCAGGCGGCACCTCTTATAATCAGAAGTTTAAAGGGCGCTTTACCCTGTCCGTGGACAATTCCAAGAATACTGCTTACCTGCAGATGAATTCCCTGCGCGCCGAAGATACAGCTGTGTATTACTGCGCCAGAGAAGGGATCTATTGGTGGGGACAGGGCACCCTCGTGACAGTGTCATCCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCCGGAGGAGGGGGAAGTGGCGGCGGAGGATCTGAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCAGCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGCAGCGGATTTGACTACTGGGGCCAAGGAACCCTGGTCAC CGTCTCGAGC CD40GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCG 116 VHCH1-CD40GCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTACAGCTT VHCH1-Fchole_PGLALA-CACCGGCTACTACATCCACTGGGTGAGGCAGGCCCCCGGCAAG DP47 VLGGCCTGGAGTGGGTGGCCAGGGTGATCCCCAACGCCGGCGGCACCAGCTACAACCAGAAGTTCAAGGGCAGGTTCACCCTGAGCGTGGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGGCATCTACTGGTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTACAGCTTCACCGGCTACTACATCCACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCAGGGTGATCCCCAACGCCGGCGGCACCAGCTACAACCAGAAGTTCAAGGGCAGGTTCACCCTGAGCGTGGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGGCATCTACTGGTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGGCCGCCGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGGGCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTGCACCCTGCCCCCCAGCAGGGACGAGCTGACCAAGAACCAGGTGAGCCTGAGCTGCGCCGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGGTGAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGGCAGCAGCCCCCTGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG +CD40 LC see above 106 pETR15390 CD40 xDP47 (4 + 2) CD40 GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 117VHCH1-CD40 GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTT VHCH1-CACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAG Fc_PGLALA-GGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAA DP47 VLCH1CCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGT ‘EE’GGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCATCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGCGCTTCTACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGGAGGACTACTTTCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTCCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACTGTGCCCAGCAGCAGCCTGGGAACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAACCCAAGAGCTGCGACGGCGGAGGCGGATCTGGCGGCGGAGGATCCGAAGTGCAGCTGGTGGAAAGTGGGGGAGGCCTGGTGCAGCCAGGGGGAAGCCTGAGACTGTCTTGTGCCGCTTCCGGCTACTCTTTTACCGGGTATTATATCCATTGGGTGCGGCAGGCTCCAGGGAAAGGCCTGGAATGGGTGGCACGCGTGATCCCTAACGCAGGCGGCACCTCTTATAATCAGAAGTTTAAAGGGCGCTTTACCCTGTCCGTGGACAATTCCAAGAATACTGCTTACCTGCAGATGAATTCCCTGCGCGCCGAAGATACAGCTGTGTATTACTGCGCCAGAGAAGGGATCTATTGGTGGGGACAGGGCACCCTCGTGACAGTGTCATCCGCTAGCACCAAGGGACCTTCCGTGTTTCCCCTGGCTCCCAGCTCCAAGTCTACCTCTGGGGGCACAGCTGCTCTGGGATGTCTGGTGGAAGATTATTTTCCTGAACCTGTGACCGTGTCATGGAACAGCGGAGCCCTGACCTCCGGGGTGCACACATTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACACAGACATATATCTGTAATGTGAATCACAAACCCTCTAATACCAAAGTGGATGAGAAAGTGGAACCTAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCATCTGTGTTTCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCAGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCGCGGGAAGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGAGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCTCAGGTGTACACCCTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACACAGAAGTCTCTGAGCCTGAGCCCTGGCGGAGGGGGAGGATCTGGGGGAGGCGGAAGTGGGGGAGGGGGTTCCGGAGGCGGAGGATCCGAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGCTGACGTTCGGCCAGGGGACCAAAGTGGAAATCAAAAGCAGCGCTTCCACCAAGGGCCCCTCAGTGTTCCCACTGGCACCATCCAGCAAGTCCACAAGCGGAGGAACCGCCGCTCTGGGCTGTCTCGTGAAAGACTACTTTCCAGAGCCAGTGACCGTGTCCTGGAATAGTGGCGCTCTGACTTCTGGCGTGCACACTTTCCCCGCAGTGCTGCAGAGTTCTGGCCTGTACTCCCTGAGTAGCGTCGTGACAGTGCCCTCCTCTAGCCTGGGCACTCAGACTTACATCTGCAATGTGAATCATAAGCCTTCCAACACAAAAGTGGACAAAAAAGTGGAACCCAAATCT TGC +CD40 see above 110 LC;,RK’ pETR15391 +DP47VHCL GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTG 118pETR15119 GGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGCAGCGGATTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACAGCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAG TCTTTCAACCGGGGCGAGTGC muCD40-28H1 (4 + 1) CD40 GAAGTGCAGCTGGTGGAAAGCGACGGCGGACTGGTGCAGCCTG 119VHCH1-CD40 GCAGATCTCTGAAGCTGCCTTGTGCCGCCAGCGGCTTCACCTT VHCH1-CAGCGACTACTACATGGCCTGGGTGCGACAGGCCCCTACCAAG FcKK_DAPG-GGACTGGAATGGGTGGCCAGCATCAGCTACGACGGCAGCAGCA 28H1 VHCCTACTACAGAGACAGCGTGAAGGGCAGATTCACCATCAGCAG pETR15732AGACAACGCCAAGAGCACCCTGTACCTGCAGATGGACAGCCTGAGAAGCGAGGACACCGCTACCTACTACTGCGGCAGACACAGCAGCTACTTCGACTACTGGGGCCAGGGCGTGATGGTCACCGTGTCTAGCGCCAAGACCACACCCCCCAGCGTGTACCCTCTGGCTCCTGGATCTGCCGCCCAGACCAACAGCATGGTCACACTGGGCTGCCTGGTGAAGGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAACAGCGGCTCTCTGTCTAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCGACCTGTACACCCTGTCCTCCAGCGTGACCGTGCCTTCCTCCACCTGGCCTTCCCAGACCGTGACATGCAACGTGGCCCACCCTGCCAGCTCCACCAAGGTGGACAAGAAAATCGTGCCCCGGGACTGCGGAGGGGGCGGTTCCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAATCTGATGGGGGCCTGGTGCAGCCCGGAAGAAGCCTGAAACTGCCCTGTGCTGCCTCTGGCTTCACATTCTCTGATTACTATATGGCTTGGGTGCGCCAGGCTCCAACAAAAGGCCTGGAATGGGTGGCATCCATCTCTTACGACGGCTCCTCCACTTACTACAGGGACTCTGTGAAGGGCCGGTTCACAATCTCCCGGGATAACGCCAAGTCTACACTGTACCTGCAGATGGATTCCCTGCGCTCCGAGGACACAGCCACATATTACTGTGGCAGGCACTCCTCCTACTTTGATTATTGGGGACAGGGCGTGATGGTCACAGTGTCCAGCGCTAAGACCACCCCCCCTAGCGTGTACCCTCTGGCCCCTGGATCTGCCGCCCAGACCAACAGCATGGTGACCCTGGGCTGCCTGGTGAAGGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAACAGCGGCAGCCTGAGCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCGACCTGTACACCCTGAGCAGCTCCGTGACCGTGCCTAGCAGCACCTGGCCCAGCCAGACAGTGACCTGCAACGTGGCCCACCCTGCCAGCAGCACCAAGGTGGACAAGAAAATCGTGCCCCGGGACTGCGGCTGCAAGCCCTGCATCTGCACCGTGCCCGAGGTGTCCAGCGTGTTCATCTTCCCACCCAAGCCCAAGGACGTGCTGACCATCACCCTGACCCCCAAAGTGACCTGCGTGGTGGTGGCCATCAGCAAGGACGACCCCGAGGTGCAGTTCTCTTGGTTTGTGGACGACGTGGAGGTGCACACAGCCCAGACAAAGCCCCGGGAGGAACAGATCAACAGCACCTTCAGAAGCGTGTCCGAGCTGCCCATCATGCACCAGGACTGGCTGAACGGCAAAGAATTCAAGTGCAGAGTGAACAGCGCCGCCTTCGGCGCCCCCATCGAGAAAACCATCAGCAAGACCAAGGGCAGACCCAAGGCCCCCCAGGTGTACACCATCCCCCCACCCAAAAAACAGATGGCCAAGGACAAGGTGTCCCTGACCTGCATGATCACCAACTTTTTCCCCGAGGACATCACCGTGGAGTGGCAGTGGAATGGCCAGCCCGCCGAGAACTACAAGAACACCCAGCCCATCATGAAGACCGACGGCAGCTACTTCGTGTACAGCAAGCTGAACGTGCAGAAGTCCAACTGGGAGGCCGGCAACACCTTCACCTGTAGCGTGCTGCACGAGGGCCTGCACAACCACCACACCGAGAAGTCCCTGAGCCACTCCCCCGGCGGCGGAGGCGGTTCCGGAGGAGGAGGATCCGGAGGAGGGGGAAGTGGCGGCGGAGGATCTGAGGTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGCAGCCTGGCGGATCTCTGAGACTGTCCTGCGCCGCCTCCGGCTTCACCTTCTCCTCCCACGCCATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGACTCTGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGGCTGGCTGGGCAACTTCGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCAG C CD40GAAGTGCAGCTGGTGGAAAGCGACGGCGGACTGGTGCAGCCTG 120 VHCH1-CD40GCAGATCTCTGAAGCTGCCTTGTGCCGCCAGCGGCTTCACCTT VHCH1-CAGCGACTACTACATGGCCTGGGTGCGACAGGCCCCTACCAAG FcDD_DAPG-GGACTGGAATGGGTGGCCAGCATCAGCTACGACGGCAGCAGCA 28H1 VLCCTACTACAGAGACAGCGTGAAGGGCAGATTCACCATCAGCAG pETR15731AGACAACGCCAAGAGCACCCTGTACCTGCAGATGGACAGCCTGAGAAGCGAGGACACCGCTACCTACTACTGCGGCAGACACAGCAGCTACTTCGACTACTGGGGCCAGGGCGTGATGGTCACCGTGTCTAGCGCCAAGACCACACCCCCCAGCGTGTACCCTCTGGCTCCTGGATCTGCCGCCCAGACCAACAGCATGGTCACACTGGGCTGCCTGGTGAAGGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAACAGCGGCTCTCTGTCTAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCGACCTGTACACCCTGTCCTCCAGCGTGACCGTGCCTTCCTCCACCTGGCCTTCCCAGACCGTGACATGCAACGTGGCCCACCCTGCCAGCTCCACCAAGGTGGACAAGAAAATCGTGCCCCGGGACTGCGGAGGGGGCGGTTCCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAATCTGATGGGGGCCTGGTGCAGCCCGGAAGAAGCCTGAAACTGCCCTGTGCTGCCTCTGGCTTCACATTCTCTGATTACTATATGGCTTGGGTGCGCCAGGCTCCAACAAAAGGCCTGGAATGGGTGGCATCCATCTCTTACGACGGCTCCTCCACTTACTACAGGGACTCTGTGAAGGGCCGGTTCACAATCTCCCGGGATAACGCCAAGTCTACACTGTACCTGCAGATGGATTCCCTGCGCTCCGAGGACACAGCCACATATTACTGTGGCAGGCACTCCTCCTACTTTGATTATTGGGGACAGGGCGTGATGGTCACAGTGTCCAGCGCTAAGACCACCCCCCCTAGCGTGTACCCTCTGGCCCCTGGATCTGCCGCCCAGACCAACAGCATGGTGACCCTGGGCTGCCTGGTGAAGGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAACAGCGGCAGCCTGAGCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCGACCTGTACACCCTGAGCAGCTCCGTGACCGTGCCTAGCAGCACCTGGCCCAGCCAGACAGTGACCTGCAACGTGGCCCACCCTGCCAGCAGCACCAAGGTGGACAAGAAAATCGTGCCCCGGGACTGCGGCTGCAAGCCCTGCATCTGCACCGTGCCCGAGGTGTCCAGCGTGTTCATCTTCCCACCCAAGCCCAAGGACGTGCTGACCATCACCCTGACCCCCAAAGTGACCTGCGTGGTGGTGGCCATCAGCAAGGACGACCCCGAGGTGCAGTTCTCTTGGTTTGTGGACGACGTGGAGGTGCACACAGCCCAGACAAAGCCCCGGGAGGAACAGATCAACAGCACCTTCAGAAGCGTGTCCGAGCTGCCCATCATGCACCAGGACTGGCTGAACGGCAAAGAATTCAAGTGCAGAGTGAACAGCGCCGCCTTCGGCGCCCCCATCGAGAAAACCATCAGCAAGACCAAGGGCAGACCCAAGGCCCCCCAGGTGTACACCATCCCCCCACCCAAAGAACAGATGGCCAAGGACAAGGTGTCCCTGACCTGCATGATCACCAACTTTTTCCCCGAGGACATCACCGTGGAGTGGCAGTGGAATGGCCAGCCCGCCGAGAACTACGACAACACCCAGCCCATCATGGACACCGACGGCAGCTACTTCGTGTACAGCGACCTGAACGTGCAGAAGTCCAACTGGGAGGCCGGCAACACCTTCACCTGTAGCGTGCTGCACGAGGGCCTGCACAACCACCACACCGAGAAGTCCCTGAGCCACAGCCCAGGCGGCGGAGGCGGATCTGGCGGAGGAGGTTCCGGAGGTGGCGGATCTGGGGGCGGTGGATCTGAGATCGTGCTGACCCAGTCTCCCGGCACCCTGAGCCTGAGCCCTGGCGAGAGAGCCACCCTGAGCTGCAGAGCCAGCCAGAGCGTGAGCCGGAGCTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCATCGGCGCCAGCACCCGGGCCACCGGCATCCCCGATAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCCAGGTGATCCCCCCCACCTTCGGCCAGG GCACCAAGGTGGAAATCAAG mu CD40light GACACTGTACTGACCCAGTCTCCTGCTTTGGCTGTGTCTCCAG 121 chainGAGAGAGGGTTACCATCTCCTGTAGGGCCAGTGACAGTGTCAG pETR13185TACACTTATGCACTGGTACCAACAGAAACCAGGACAGCAACCCAAACTCCTCATCTATCTAGCATCACACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATTGATCCTGTGGAGGCTGATGACACTGCAACCTATTACTGTCAGCAGAGTTGGAATGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAATTGAAACGTGCCGATGCTGCACCAACTGTATCGATTTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT mu CD40- 28H1 (4 + 2) CD40GAAGTGCAGCTGGTGGAATCCGACGGCGGACTGGTGCAGCCTG 122 VHCH1-CD40GCAGATCTCTGAAGCTGCCTTGTGCCGCCTCCGGCTTCACCTT VHCH1-CTCCGACTACTACATGGCCTGGGTGCGACAGGCCCCTACCAAG Fc_DAPG-GGACTGGAATGGGTGGCCTCCATCTCCTACGACGGCTCCTCCA 28H1 VLCH1CCTACTACCGGGACTCTGTGAAGGGCCGGTTCACCATCTCTCG pETR15744GGACAACGCCAAGTCCACCCTGTACCTGCAGATGGACTCCCTGCGGAGCGAGGACACCGCTACCTACTACTGCGGCAGACACTCCTCCTACTTCGACTACTGGGGCCAGGGCGTGATGGTCACCGTGTCCTCTGCTAAGACCACCCCCCCCTCCGTGTACCCTCTGGCTCCTGGATCTGCCGCCCAGACCAACTCCATGGTCACCCTGGGCTGTCTGGTGGAAGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAACTCCGGCTCTCTGTCCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCGACCTGTACACCCTGAGCAGCTCCGTGACCGTGCCTAGCAGCACCTGGCCCAGCCAGACAGTGACCTGCAACGTGGCCCACCCTGCCAGCAGCACCAAGGTGGACGAGAAAATCGTGCCCCGGGACTGCGGCGGTGGAGGTTCCGGAGGCGGCGGATCCGAGGTGCAGCTGGTGGAAAGTGATGGGGGCCTGGTGCAGCCCGGAAGAAGCCTGAAACTGCCCTGCGCCGCTTCTGGCTTTACCTTTAGCGATTACTATATGGCTTGGGTGCGCCAGGCTCCAACAAAAGGCCTGGAATGGGTGGCATCTATCAGCTACGATGGCAGCAGCACCTACTATAGAGACAGCGTGAAGGGGAGATTCACCATCAGCAGAGATAACGCTAAGAGCACACTGTACCTGCAGATGGATAGCCTGAGATCCGAGGATACCGCCACATATTACTGTGGCCGGCACAGCAGCTACTTTGATTATTGGGGACAGGGCGTGATGGTCACAGTGTCTAGCGCTAAGACTACCCCTCCTAGCGTGTACCCCCTGGCACCAGGTTCCGCTGCTCAGACCAACAGCATGGTCACACTGGGATGCCTGGTGGAAGGATATTTTCCTGAACCCGTGACAGTGACATGGAATAGCGGCTCCCTGTCTAGCGGAGTGCATACCTTTCCAGCTGTGCTGCAGAGCGATCTGTATACACTGAGCAGCTCTGTGACAGTGCCTTCCAGCACCTGGCCCAGCCAGACAGTGACCTGTAATGTGGCTCATCCCGCCTCTAGCACCAAAGTGGATGAGAAAATCGTGCCCCGGGACTGCGGCTGCAAGCCCTGTATCTGTACCGTGCCCGAGGTGTCCTCCGTGTTCATCTTCCCACCTAAGCCCAAGGACGTGCTGACAATCACCCTGACCCCCAAAGTGACCTGCGTGGTGGTGGCCATCTCCAAGGACGATCCCGAGGTGCAGTTCAGTTGGTTCGTGGACGACGTGGAAGTGCACACAGCCCAGACAAAGCCCAGAGAGGAACAGATCAACTCCACCTTCAGAAGCGTGTCCGAGCTGCCCATCATGCACCAGGACTGGCTGAACGGCAAAGAATTCAAGTGCAGAGTGAACTCCGCCGCCTTTGGCGCCCCTATCGAAAAGACCATCTCCAAGACCAAGGGCAGACCCAAGGCCCCCCAGGTGTACACAATCCCCCCACCCAAAGAACAGATGGCCAAGGACAAGGTGTCCCTGACCTGCATGATCACCAACTTTTTCCCAGAGGACATCACCGTGGAATGGCAGTGGAACGGCCAGCCCGCCGAGAACTACAAGAACACCCAGCCCATCATGGACACCGACGGCTCCTACTTCGTGTACTCCAAGCTGAACGTGCAGAAGTCCAACTGGGAGGCCGGCAACACCTTCACCTGTTCCGTGCTGCACGAGGGCCTGCACAACCACCACACCGAGAAGTCCCTGTCCCACTCCCCTGGAAAAGGCGGAGGCGGATCTGGTGGCGGAGGATCTGGCGGTGGTGGTTCCGGAGGCGGTGGATCTGAGATCGTGCTGACCCAGTCTCCCGGCACCCTGTCACTGTCTCCAGGCGAGAGAGCCACCCTGTCCTGCAGAGCCTCTCAGTCCGTGTCCCGGTCTTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCTCCCCGGCTGCTGATCATCGGAGCTTCTACCAGAGCCACCGGCATCCCCGACAGATTCTCCGGCTCTGGCTCTGGCACCGACTTCACCCTGACCATCTCTCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCCAAGTGATCCCCCCCACCTTTGGCCAGGGCACCAAGGTGGAAATCAAGTCCAGCGCTAAGACCACCCCCCCCTCCGTGTATCCTCTGGCCCCTGGATCTGCCGCCCAGACCAACTCCATGGTCACCCTGGGCTGCCTCGTGAAGGGCTACTTCCCTGAGCCTGTGACCGTGACCTGGAACTCCGGCTCCCTGTCTAGCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCGACCTGTACACCCTGAGCAGCTCCGTGACCGTGCCTTCCTCCACCTGGCCTTCCCAGACCGTGACATGCAACGTGGCCCACCCTGCCAGCTCCACAAAGGTGGACAAGAAAATCGTGCCCCGGGACTGC 28H1 VHCLGAAGTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGCAGCCTG 123 (mu)GCGGATCTCTGAGACTGTCCTGCGCCGCCTCCGGCTTCACCTT pETR15650CTCCTCCCACGCCATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGACTCTGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGGCTGGCTGGGCAACTTCGACTACTGGGGACAGGGCACCCTGGTCACCGTGTCCAGCGCTTCTGATGCCGCCCCTACCGTATCGATTTTCCCACCCTCCAGCGAGCAGCTGACAAGCGGCGGAGCTAGCGTCGTGTGCTTCCTGAACAACTTCTACCCCAAGGACATCAACGTGAAGTGGAAGATCGACGGCAGCGAGCGGCAGAACGGCGTGCTGAATAGCTGGACCGACCAGGACAGCAAGGACTCCACCTACAGCATGAGCAGCACCCTGACCCTGACCAAGGACGAGTACGAGCGGCACAACAGCTACACATGCGAGGCCACCCACAAGACCAGCACCAGCCCCATCGTG AAGTCCTTCAACCGGAACGAGTGC muCD40 light GACACTGTACTGACCCAGTCTCCTGCTTTGGCTGTGTCTCCAG 124 chain; ‘RK’GAGAGAGGGTTACCATCTCCTGTAGGGCCAGTGACAGTGTCAG pETR15649TACACTTATGCACTGGTACCAACAGAAACCAGGACAGCAACCCAAACTCCTCATCTATCTAGCATCACACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATTGATCCTGTGGAGGCTGATGACACTGCAACCTATTACTGTCAGCAGAGTTGGAATGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAATTGAAACGTGCCGATGCTGCACCAACTGTATCGATTTTCCCACCATCCAGTCGGAAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT mu CD40-DP47 (4 + 1) CD40GAAGTGCAGCTGGTGGAAAGCGACGGCGGACTGGTGCAGCCTG 125 VHCH1-CD40GCAGATCTCTGAAGCTGCCTTGTGCCGCCAGCGGCTTCACCTT VHCH1-CAGCGACTACTACATGGCCTGGGTGCGACAGGCCCCTACCAAG FcKK_DAPG-GGACTGGAATGGGTGGCCAGCATCAGCTACGACGGCAGCAGCA DP47 VHCCTACTACAGAGACAGCGTGAAGGGCAGATTCACCATCAGCAG pETR15734AGACAACGCCAAGAGCACCCTGTACCTGCAGATGGACAGCCTGAGAAGCGAGGACACCGCTACCTACTACTGCGGCAGACACAGCAGCTACTTCGACTACTGGGGCCAGGGCGTGATGGTCACCGTGTCTAGCGCCAAGACCACACCCCCCAGCGTGTACCCTCTGGCTCCTGGATCTGCCGCCCAGACCAACAGCATGGTCACACTGGGCTGCCTGGTGAAGGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAACAGCGGCTCTCTGTCTAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCGACCTGTACACCCTGTCCTCCAGCGTGACCGTGCCTTCCTCCACCTGGCCTTCCCAGACCGTGACATGCAACGTGGCCCACCCTGCCAGCTCCACCAAGGTGGACAAGAAAATCGTGCCCCGGGACTGCGGAGGGGGCGGTTCCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAATCTGATGGGGGCCTGGTGCAGCCCGGAAGAAGCCTGAAACTGCCCTGTGCTGCCTCTGGCTTCACATTCTCTGATTACTATATGGCTTGGGTGCGCCAGGCTCCAACAAAAGGCCTGGAATGGGTGGCATCCATCTCTTACGACGGCTCCTCCACTTACTACAGGGACTCTGTGAAGGGCCGGTTCACAATCTCCCGGGATAACGCCAAGTCTACACTGTACCTGCAGATGGATTCCCTGCGCTCCGAGGACACAGCCACATATTACTGTGGCAGGCACTCCTCCTACTTTGATTATTGGGGACAGGGCGTGATGGTCACAGTGTCCAGCGCTAAGACCACCCCCCCTAGCGTGTACCCTCTGGCCCCTGGATCTGCCGCCCAGACCAACAGCATGGTGACCCTGGGCTGCCTGGTGAAGGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAACAGCGGCAGCCTGAGCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCGACCTGTACACCCTGAGCAGCTCCGTGACCGTGCCTAGCAGCACCTGGCCCAGCCAGACAGTGACCTGCAACGTGGCCCACCCTGCCAGCAGCACCAAGGTGGACAAGAAAATCGTGCCCCGGGACTGCGGCTGCAAGCCCTGCATCTGCACCGTGCCCGAGGTGTCCAGCGTGTTCATCTTCCCACCCAAGCCCAAGGACGTGCTGACCATCACCCTGACCCCCAAAGTGACCTGCGTGGTGGTGGCCATCAGCAAGGACGACCCCGAGGTGCAGTTCTCTTGGTTTGTGGACGACGTGGAGGTGCACACAGCCCAGACAAAGCCCCGGGAGGAACAGATCAACAGCACCTTCAGAAGCGTGTCCGAGCTGCCCATCATGCACCAGGACTGGCTGAACGGCAAAGAATTCAAGTGCAGAGTGAACAGCGCCGCCTTCGGCGCCCCCATCGAGAAAACCATCAGCAAGACCAAGGGCAGACCCAAGGCCCCCCAGGTGTACACCATCCCCCCACCCAAAAAACAGATGGCCAAGGACAAGGTGTCCCTGACCTGCATGATCACCAACTTTTTCCCCGAGGACATCACCGTGGAGTGGCAGTGGAATGGCCAGCCCGCCGAGAACTACAAGAACACCCAGCCCATCATGAAGACCGACGGCAGCTACTTCGTGTACAGCAAGCTGAACGTGCAGAAGTCCAACTGGGAGGCCGGCAACACCTTCACCTGTAGCGTGCTGCACGAGGGCCTGCACAACCACCACACCGAGAAGTCCCTGAGCCACTCCCCCGGCGGCGGAGGCGGTTCCGGAGGAGGAGGATCCGGAGGAGGGGGAAGTGGCGGCGGAGGATCTGAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCAGCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGCAGCGGATTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGC CD40GAAGTGCAGCTGGTGGAAAGCGACGGCGGACTGGTGCAGCCTG 126 VHCH1-CD40GCAGATCTCTGAAGCTGCCTTGTGCCGCCAGCGGCTTCACCTT VHCH1-CAGCGACTACTACATGGCCTGGGTGCGACAGGCCCCTACCAAG FcDD_DAPG-GGACTGGAATGGGTGGCCAGCATCAGCTACGACGGCAGCAGCA DP47 VLCCTACTACAGAGACAGCGTGAAGGGCAGATTCACCATCAGCAG pETR15733AGACAACGCCAAGAGCACCCTGTACCTGCAGATGGACAGCCTGAGAAGCGAGGACACCGCTACCTACTACTGCGGCAGACACAGCAGCTACTTCGACTACTGGGGCCAGGGCGTGATGGTCACCGTGTCTAGCGCCAAGACCACACCCCCCAGCGTGTACCCTCTGGCTCCTGGATCTGCCGCCCAGACCAACAGCATGGTCACACTGGGCTGCCTGGTGAAGGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAACAGCGGCTCTCTGTCTAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCGACCTGTACACCCTGTCCTCCAGCGTGACCGTGCCTTCCTCCACCTGGCCTTCCCAGACCGTGACATGCAACGTGGCCCACCCTGCCAGCTCCACCAAGGTGGACAAGAAAATCGTGCCCCGGGACTGCGGAGGGGGCGGTTCCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAATCTGATGGGGGCCTGGTGCAGCCCGGAAGAAGCCTGAAACTGCCCTGTGCTGCCTCTGGCTTCACATTCTCTGATTACTATATGGCTTGGGTGCGCCAGGCTCCAACAAAAGGCCTGGAATGGGTGGCATCCATCTCTTACGACGGCTCCTCCACTTACTACAGGGACTCTGTGAAGGGCCGGTTCACAATCTCCCGGGATAACGCCAAGTCTACACTGTACCTGCAGATGGATTCCCTGCGCTCCGAGGACACAGCCACATATTACTGTGGCAGGCACTCCTCCTACTTTGATTATTGGGGACAGGGCGTGATGGTCACAGTGTCCAGCGCTAAGACCACCCCCCCTAGCGTGTACCCTCTGGCCCCTGGATCTGCCGCCCAGACCAACAGCATGGTGACCCTGGGCTGCCTGGTGAAGGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAACAGCGGCAGCCTGAGCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCGACCTGTACACCCTGAGCAGCTCCGTGACCGTGCCTAGCAGCACCTGGCCCAGCCAGACAGTGACCTGCAACGTGGCCCACCCTGCCAGCAGCACCAAGGTGGACAAGAAAATCGTGCCCCGGGACTGCGGCTGCAAGCCCTGCATCTGCACCGTGCCCGAGGTGTCCAGCGTGTTCATCTTCCCACCCAAGCCCAAGGACGTGCTGACCATCACCCTGACCCCCAAAGTGACCTGCGTGGTGGTGGCCATCAGCAAGGACGACCCCGAGGTGCAGTTCTCTTGGTTTGTGGACGACGTGGAGGTGCACACAGCCCAGACAAAGCCCCGGGAGGAACAGATCAACAGCACCTTCAGAAGCGTGTCCGAGCTGCCCATCATGCACCAGGACTGGCTGAACGGCAAAGAATTCAAGTGCAGAGTGAACAGCGCCGCCTTCGGCGCCCCCATCGAGAAAACCATCAGCAAGACCAAGGGCAGACCCAAGGCCCCCCAGGTGTACACCATCCCCCCACCCAAAGAACAGATGGCCAAGGACAAGGTGTCCCTGACCTGCATGATCACCAACTTTTTCCCCGAGGACATCACCGTGGAGTGGCAGTGGAATGGCCAGCCCGCCGAGAACTACGACAACACCCAGCCCATCATGGACACCGACGGCAGCTACTTCGTGTACAGCGACCTGAACGTGCAGAAGTCCAACTGGGAGGCCGGCAACACCTTCACCTGTAGCGTGCTGCACGAGGGCCTGCACAACCACCACACCGAGAAGTCCCTGAGCCACAGCCCAGGCGGCGGAGGCGGATCTGGCGGAGGAGGTTCCGGAGGCGGCGGAAGCGGAGGGGGAGGCTCTGAAATTGTGCTGACCCAGAGCCCCGGCACCCTGTCACTGTCTCCAGGCGAAAGAGCCACCCTGAGCTGCAGAGCCAGCCAGAGCGTGTCCAGCTCTTACCTGGCCTGGTATCAGCAGAAGCCCGGACAGGCCCCCAGACTGCTGATCTACGGCGCCTCTTCTAGAGCCACCGGCATCCCCGATAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACAATCAGCAGACTGGAACCCGAGGACTTTGCCGTGTATTACTGCCAGCAGTACGGCAGCAGCCCCCTGACCTTTGGCCAGG GCACCAAGGTGGAAATCAAA +muCD40 see above 121 light chain mu CD40 x DP47 (4 + 2) CD40GAAGTGCAGCTGGTGGAATCCGACGGCGGACTGGTGCAGCCTG 127 VHCH1-CD40GCAGATCTCTGAAGCTGCCTTGTGCCGCCTCCGGCTTCACCTT VHCH1-CTCCGACTACTACATGGCCTGGGTGCGACAGGCCCCTACCAAG Fc_DAPG-GGACTGGAATGGGTGGCCTCCATCTCCTACGACGGCTCCTCCA 28H1 VLCH1CCTACTACCGGGACTCTGTGAAGGGCCGGTTCACCATCTCTCG ‘EE’GGACAACGCCAAGTCCACCCTGTACCTGCAGATGGACTCCCTG pETR15748CGGAGCGAGGACACCGCTACCTACTACTGCGGCAGACACTCCTCCTACTTCGACTACTGGGGCCAGGGCGTGATGGTCACCGTGTCCTCTGCTAAGACCACCCCCCCCTCCGTGTACCCTCTGGCTCCTGGATCTGCCGCCCAGACCAACTCCATGGTCACCCTGGGCTGTCTGGTGGAAGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAACTCCGGCTCTCTGTCCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCGACCTGTACACCCTGAGCAGCTCCGTGACCGTGCCTAGCAGCACCTGGCCCAGCCAGACAGTGACCTGCAACGTGGCCCACCCTGCCAGCAGCACCAAGGTGGACGAGAAAATCGTGCCCCGGGACTGCGGCGGTGGAGGTTCCGGAGGCGGCGGATCCGAGGTGCAGCTGGTGGAAAGTGATGGGGGCCTGGTGCAGCCCGGAAGAAGCCTGAAACTGCCCTGCGCCGCTTCTGGCTTTACCTTTAGCGATTACTATATGGCTTGGGTGCGCCAGGCTCCAACAAAAGGCCTGGAATGGGTGGCATCTATCAGCTACGATGGCAGCAGCACCTACTATAGAGACAGCGTGAAGGGGAGATTCACCATCAGCAGAGATAACGCTAAGAGCACACTGTACCTGCAGATGGATAGCCTGAGATCCGAGGATACCGCCACATATTACTGTGGCCGGCACAGCAGCTACTTTGATTATTGGGGACAGGGCGTGATGGTCACAGTGTCTAGCGCTAAGACTACCCCTCCTAGCGTGTACCCCCTGGCACCAGGTTCCGCTGCTCAGACCAACAGCATGGTCACACTGGGATGCCTGGTGGAAGGATATTTTCCTGAACCCGTGACAGTGACATGGAATAGCGGCTCCCTGTCTAGCGGAGTGCATACCTTTCCAGCTGTGCTGCAGAGCGATCTGTATACACTGAGCAGCTCTGTGACAGTGCCTTCCAGCACCTGGCCCAGCCAGACAGTGACCTGTAATGTGGCTCATCCCGCCTCTAGCACCAAAGTGGATGAGAAAATCGTGCCCCGGGACTGCGGCTGCAAGCCCTGTATCTGTACCGTGCCCGAGGTGTCCTCCGTGTTCATCTTCCCACCTAAGCCCAAGGACGTGCTGACAATCACCCTGACCCCCAAAGTGACCTGCGTGGTGGTGGCCATCTCCAAGGACGATCCCGAGGTGCAGTTCAGTTGGTTCGTGGACGACGTGGAAGTGCACACAGCCCAGACAAAGCCCAGAGAGGAACAGATCAACTCCACCTTCAGAAGCGTGTCCGAGCTGCCCATCATGCACCAGGACTGGCTGAACGGCAAAGAATTCAAGTGCAGAGTGAACTCCGCCGCCTTTGGCGCCCCTATCGAAAAGACCATCTCCAAGACCAAGGGCAGACCCAAGGCCCCCCAGGTGTACACAATCCCCCCACCCAAAGAACAGATGGCCAAGGACAAGGTGTCCCTGACCTGCATGATCACCAACTTTTTCCCAGAGGACATCACCGTGGAATGGCAGTGGAACGGCCAGCCCGCCGAGAACTACAAGAACACCCAGCCCATCATGGACACCGACGGCTCCTACTTCGTGTACTCCAAGCTGAACGTGCAGAAGTCCAACTGGGAGGCCGGCAACACCTTCACCTGTTCCGTGCTGCACGAGGGCCTGCACAACCACCACACCGAGAAGTCCCTGTCCCACTCCCCTGGAAAAGGCGGAGGCGGATCTGGTGGCGGAGGATCTGGCGGTGGTGGTTCCGGAGGCGGAGGATCCGAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGCTGACGTTCGGCCAGGGGACCAAAGTGGAAATCAAAAGCAGCGCTAAGACCACCCCCCCCTCCGTGTATCCTCTGGCCCCTGGATCTGCCGCCCAGACCAACTCCATGGTCACCCTGGGCTGCCTCGTGAAGGGCTACTTCCCTGAGCCTGTGACCGTGACCTGGAACTCCGGCTCCCTGTCTAGCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCGACCTGTACACCCTGAGCAGCTCCGTGACCGTGCCTTCCTCCACCTGGCCTTCCCAGACCGTGACATGCAACGTGGCCCACCCTGCCAGCTCCACAAAGGTGGACAAGAAAATCGTGCCCCGGGACTGC +mu CD40 see above 124 light chain;,RK’ DP47 VHCL GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTG 128 (mu)GGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTT pETR15652TAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGCAGCGGATTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGCGCTTCTGATGCCGCCCCTACCGTATCGATTTTCCCACCCTCCAGCGAGCAGCTGACAAGCGGCGGAGCTAGCGTCGTGTGCTTCCTGAACAACTTCTACCCCAAGGACATCAACGTGAAGTGGAAGATCGACGGCAGCGAGCGGCAGAACGGCGTGCTGAATAGCTGGACCGACCAGGACAGCAAGGACTCCACCTACAGCATGAGCAGCACCCTGACCCTGACCAAGGACGAGTACGAGCGGCACAACAGCTACACATGCGAGGCCACCCACAAGACCAGCACCAGCCCCATCGTGAAG TCCTTCAACCGGAACGAGTGC

1.2 Production of Bispecific Antigen Binding Molecules Targeting CD40and Fibroblast Activation Protein (FAP)

The molecules were produced by co-transfecting either HEK293-EBNA cellsgrowing in suspension with the mammalian expression vectors usingpolyethylenimine (PEI) or co-transfecting CHO K1 cells growing insuspension with the mammalian expression using eviFECT. The cells weretransfected with the corresponding expression vectors.

For production in HEK293 EBNA cells HEK293 EBNA cells were cultivated insuspension serum free in Excell culture medium containing 6 mML-glutamine and 250 mg/L G418. For the production in 600 mL tubespinflasks (max. working volume 400 mL) 600 million HEK293 EBNA cells wereseeded 24 hours before transfection. For transfection 800 million cellswere centrifuged for 5 min at 210×g and supernatant was replaced by 20mL pre-warmed CD CHO medium. Expression vectors were mixed in 20 mL CDCHO medium to a final amount of 400 μg DNA. After addition of 1080 μgPEI solution (2.7 μg/mL) the mixture was vortexed for 15 s andsubsequently incubated for 10 min at room temperature. Afterwards cellswere mixed with the DNA/PEI solution, transferred to a 600 mL tubespinflask and incubated for 3 hours at 37° C. in an incubator with a 5% CO2atmosphere. After incubation, 360 mL Excell medium, containing 6 mML-glutamine, 5 g/L Pepsoy and 1.25 mM VPA, was added and cells werecultivated for 24 hours. One day after transfection 12% Feed 7 and 3 g/1Glucose were added. After 7 days the cultivation supernatant wascollected for purification by centrifugation for 60 min at 2500×g (Sigma8K centrifuge). The solution was sterile filtered (0.22 μm filter) andsodium azide was added to a final concentration of 0.01% w/v and kept at4° C.

For production in HEK293 EBNA cells HEK293 EBNA in suspension-adaptedCHO K1 cells (adapted to serum-free growth in suspension culture) thecells were grown in eviGrow medium (evitria AG, Switzerland), achemically defined, animal-component free, serum-free medium andtransfected with eviFect (evitria AG, Switzerland). After transfectionthe cells were kept in eviMake (evitria AG, Switzerland), a chemicallydefined, animal-component free, serum-free medium, at 37° C. and 5% CO₂for 7 days.

After 7 days the cultivation supernatant was collected for purificationby centrifugation for 45 min at maximum speed in a Rotanta 460 RC. Thesolution was sterile filtered (0.22 μm filter) and kept at 4° C. Theconcentration of the molecules in the culture medium was eitherdetermined by Protein A-HPLC or Protein A-Bio-Layer Interferometry(BLI).

The secreted protein was purified from cell culture supernatants byaffinity chromatography using Protein A affinity chromatography,followed by a size exclusion chromatographic step. For affinitychromatography supernatant was loaded on a HiTrap MabSelect SuRe column(Column Volume (CV)=5 mL, GE Healthcare) equilibrated with 25 ml 20 mMsodium phosphate, 20 mM sodium citrate, pH 7.5. Unbound protein wasremoved by washing with at least 10 CV 20 mM sodium phosphate, 20 mMsodium citrate, pH 7.5 and target protein was eluted in 6 CV 20 mMsodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3.0. Proteinsolution was neutralized by adding 1/10 of 0.5 M sodium phosphate, pH8.0. The target protein was concentrated and filtrated prior loading ona HiLoad XK16/60 Superdex 200 column (GE Healthcare) equilibrated with20 mM histidine, 140 mM sodium chloride, pH 6.0, 0.01% Tween20. Theprotein concentration of purified protein sample was determined bymeasuring the optical density (OD) at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence.

Purity and molecular weight of the molecule after the final purificationstep were analyzed by CE-SDS analyses in the presence and absence of areducing agent. The Caliper LabChip GXII system (Caliper Lifescience)was used according to the manufacturer's instruction.

The aggregate content of the molecule was analyzed using a TSKgel G3000SW XL analytical size-exclusion column (Tosoh) in 25 mM potassiumphosphate, 125 mM sodium chloride, 200 mM L-arginine monohydrocloride,0.02% (w/v) NaN₃, pH 6.7 running buffer at 25° C.

TABLE 5 Production yield and quality of bispecific CD40 antigen bindingmolecules Concen- Yield tration Monomer HMW LMW Construct [mg/L] [mg/ml][%] [%] [%] huCD40-28H1; 4 + 1 48.2 8.9 97.8 1.2 1.0 huCD40-28H1; 4 + 248.1 9.2 99.7 0.3 — huCD40-28H1; 2 + 1 10.1 2.0 99.5 0.5 — huCD40-28H1;2 + 2 26.7 2.0 100.0 — — huCD40-DP47; 4 + 1 15.5 2.3 98.7 1.3 —huCD40-DP47; 4 + 2 33.8 3.6 96.8 3.2 — huCD40 IgG 190.3 11.8 99.8 0.2 —muCD40-28H1; 4 + 1 3.4 0.5 92.4 7.6 — muCD40-28H1; 4 + 2 3.9 1.6 98.90.4 0.7 muCD40 IgG 19.8 5.0 97.8 — 2.2 muCD40-DP47; 4 + 1 2.2 0.3 90.69.4 — muCD40-DP47; 4 + 2 1.5 0.6 86.5 7.8 5.7

1.3 Generation of Further Bispecific Antigen Binding Molecules TargetingCD40 and Fibroblast Activation Protein (FAP)

In analogy to Example 1.1, different types of constructs of bispecificCD40-FAP antibodies have been prepared. For example, a bispecificantibody consisting of one CD40 binding moiety combined with one FAPbinding moiety was prepared (FIG. 15A). Because the CrossMab technologyas described in WO 2010/145792 A1 was used to ensure correct light chainpairing, the format is called 1+1 crossmab. Another 2+1 format called“head-to-tail” was prepared wherein a CD40 binding Fab is fused to theN-terminus of a FAP binding Fab (FIG. 15B) and a further 2+1 formatconsisting of two CD40 binding moieties combined with either one FAPbinding crossfab at the C-terminus of an Fc (FIG. 1E) was produced.Furthermore, a 4+1 format consisting of four CD40 binding moietiescombined with one FAP binding crossfab at the C-terminus of an Fc (FIG.15C) was prepared. In all these constructs, the variable heavy and lightchain domains of the anti CD40 binder correspond to the CD40 binder asdescribed in WO 2006/128103 (SEQ ID NO:10 and SEQ ID NO:16 of saiddocument). The generation and preparation of FAP binder 28H1 isdescribed in WO 2012/020006 A2, which is incorporated herein byreference. To generate the 1+1, 2+1 and 4+1 molecules the knob-into-holetechnology was used to achieve heterodimerization. The S354C/T366Wmutations have been introduced in the first heavy chain HC1 (Fc knobheavy chain) and the Y349C/T366S/L368A/Y407V mutations are introduced inthe second heavy chain HC2 (Fc hole heavy chain). Furthermore, theCrossMab technology as described in WO 2010/145792 A1 ensures correctlight chain pairing. Independent of the bispecific format, in all casesan effector silent Fc (P329G; L234, 234A) has been used to abrogatebinding to Fcγ receptors according to the method described in WO2012/130831 Al. Amino acid Sequences of the bispecific molecules areshown in Table 6.

All genes are transiently expressed under control of a chimeric MPSVpromoter consisting of the MPSV core promoter combined with the CMVpromoter enhancer fragment. The expression vector also contains the oriPregion for episomal replication in EBNA (Epstein Barr Virus NuclearAntigen) containing host cells.

TABLE 6 Amino acid sequences of the bispecific antigen binding moleculesSeq ID Construct Sequence No P1AE0192 CD40 x FAP (28H1) (1 + 1) crossmab28H1 light chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 162 crossVHCL GLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC 28H1EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQ 163 (VLCH1)_FCknob_PGLALAAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG CD40 (VHCH1EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 164 charged)_Fchole_PGLALAGLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NRFTQKSLSLSPG CD40 lightchain DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 165 (charged)KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0408 CD40 x FAP(28H1) (2 + 1) head to tail 28H1 light chainEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 162 cross VHCLGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC CD40 light chainDIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 165 (charged)KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC CD40 (VHCH1EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 166 charged)_28H1GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL (VLCH1)_FCknob_PGLALARAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG CD40(VHCH1 EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 164charged)_Fchole_PGLALA GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NRFTQKSLSLSPGK CD40 x FAP(28H1) (2 + 1) C-terminal crossfab fusion 28H1 light chainEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 162 cross VHCLGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC CD40 light chainDIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 165 (charged)KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC CD40 (VHCH1EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 167charged)_Fcknob_PGLALA_28H1 GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL(VLCH1) RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC CD40 (VHCH1EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 168 charged)_Fchole_PGLALAGLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0637 CD40x FAP (28H1) (4 + 1) C-terminal crossfab fusion 28H1 light chainEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 162 cross VHCLGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC CD40 light chainDIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 165 (charged)KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC CD40 (VHCH1EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 169 charged_CD40GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL (VHCH1RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS charged)-Fcknob_PGLALA_28H1KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ (VLCH1)SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGKGLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS C CD40 (VHCH1EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 170 charged_CD40GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL (VHCH1RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS charged)-Fchole_PGLALAKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGKGLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG

Expression of the Bispecific Antibodies

Antibodies were expressed by transient transfection of HEK cells grownin suspension with expression vectors encoding the 4 different peptidechains. Transfection into HEK293-F cells (Invitrogen) was performedaccording to the cell supplier's instructions using Maxiprep (Qiagen)preparations of the antibody vectors, F17 medium (Invitrogen, USA),Peipro (Polyscience Europe GmbH) and an initial cell density of 1-2million viable cells/ml in serum free FreeStyle 293 expression medium(Invitrogen). Cell culture supernatants were harvested after 7 days ofcultivation in shake flasks or stirred fermenters by centrifugation at14000 g for 30 minutes and filtered through a 0.22 μm filter.

Purification of the Bispecific Antibodies

Antibodies were purified from cell culture supernatants by affinitychromatography using MabSelectSure-Sepharose™ (GE Healthcare, Sweden)chromatography. Briefly, sterile filtered cell culture supernatants werecaptured on a MabSelect SuRe resin equilibrated with PBS buffer (10 mMNa₂HPO₄, 1 mM KH₂PO₄, 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed withequilibration buffer and eluted with 25 mM cirate, pH 3.0. Afterneutralization with 1 M Tris pH 9.0, aggregated protein was separatedfrom monomeric antibody species by size exclusion chromatography(Superdex 200, GE Healthcare) in 20 mM histidine, 140 mM NaCl, pH 6.0.Monomeric protein fractions were pooled, concentrated if required usinge.g. a MILLIPORE Amicon Ultra (30KD MWCO) centrifugal concentrator andstored at −80° C. Sample aliquots were used for subsequent analyticalcharacterization e.g. by CE-SDS, size exclusion chromatography, massspectrometry and endotoxin determination.

1.4 Characterization of Bispecific Constructs Targeting CD40 and FAP1.4.1 Binding to Human or Mouse FAP-Expressing Murine Fibroblast Cells

The binding to cell surface FAP was tested using human fibroblastactivating protein (huFAP) expressing cells NIH/3T3-huFAP clone 19 ormouse fibroblast activating protein (mFAP) expressing cells NIH/3T3-mFAPclone 26. NIH/3T3-huFAP clone 19 and NIH/3T3-mFAP clone 26 weregenerated by the transfection of the mouse embryonic fibroblast NIH/3T3cell line (ATCC CRL-1658) with the expression vector pETR4921 to expressunder 1.5 μg/mL Puromycin selection huFAP or mFAP, respectively. NIH/3T3wildtype (wt) cells that were not transfected with FAP and that do notexpress FAP were used as a negative control.

NIH/3T3-huFAP, NIH/3T3-mFAP or NIH/3T3-wt cells were cultured with 1×Dulbecco's Modified Eagle's Medium (DMEM) (gibco, Cat. No. 42430-025)supplemented with 10% Fetal Bovine Serum (FBS) (life technologies, Cat.No. 16140, Lot No. 1797306A). For the NIH/3T3-huFAP and NIH/3T3-mFAPcells 1.5 μg/mL Puromycin (gibco, Cat. No. A11138-03) was added to themedium for selection of FAP-expressing cells. NIH/3T3 cells were removedfrom the plate by using enzyme-free Cell Dissociation Buffer (gibco,Cat. No. 13151014). 0.3×10⁵ NIH/3T3-huFAP clone 19, NIH/3T3-mFAP clone26 or NIH/3T3-wt were added in 200 μl of 1× DMEM 10% FBS to each well ofa round-bottom 96-well plate (greiner bio-one, cellstar, Cat. No.650185). Plates were centrifuged 5 minutes at 1700 rpm and supernatantswere flicked off. Cells were washed once with 200 μL of 4° C. cold FACSbuffer (eBioscience, Cat. No. 00-4222-26). All samples were resuspendedin 50 μL/well of 4° C. cold FACS buffer containing the bispecificantigen binding molecules (primary antibody) or isotype control antibodyDP47 at the indicated range of concentrations (in duplicates) andincubated for 120 minutes at 4 ° C. Afterwards the cells were washedthree times with 200 μL 4° C. cold FACS buffer. Cells were furtherstained with 25 μL/well of 4 ° C. cold secondary antibody solution (1:50dilution of secondary antibody) containing R-Phycoerythrin (PE)conjugated AffiniPure F(ab′)₂ Fragment Goat Anti-Human IgG, Fcγ FragmentSpecific (Jackson ImmunoResearch, Cat. No. 109-116-098) and incubatedfor 60 minutes at 4 ° C. in the dark. Cells were washed with 200 μl FACSbuffer and resuspended in 85 μL/well FACS-buffer containing 0.2 μg/mLDAPI (Roche, Cat. No. 10236276001) and acquired the same day using5-laser LSR-Fortessa (BD Bioscience with DIVA software). Data analysiswas performed using the FlowJo version 10 software (FlowJo LLC).

As shown in FIG. 2A, FIG. 2B and FIG. 17, the bispecific antibodiesmonovalent or bivalent for FAP bind to human and mouse FAP-expressingtarget cells. Therefore, only FAP-targeted anti-CD40 antigen bindingmolecules show direct tumor-targeting properties. The bivalent FAPconstructs with C-terminal FAP binding domains bind stronger than themonovalent construct with C-terminal FAP binding domain explained by again of avidity in the bivalent relative to the monovalent FAP format.The strongest FAP binding was observed for the 1+1 format (P1AE0192). Nobinding of the FAP-targeted antibodies to the NIH/3T3-wt cells wasdetected. The EC₅₀ values as measured for different bispecificantibodies are shown in Table 7 below.

TABLE 7 Human FAP binding characterization of 28H1 in differentbispecific antibody formats Molecule EC₅₀ [nM] P1AD4470 CD40 IgG1 PGLALAn/a P1AE0637 CD40 × FAP 4 + 1 with C- 10.46 terminal crossfab P1AD4453CD40 × FAP 4 + 1 47.14 P1AD4574 CD40 × DP47 4 + 1 n/a P1AD4455 CD40 ×FAP 4 + 2  3.64 P1AD4465 CD40 × DP47 4 + 2 n/a P1AA9641 CD40 × FAP 2 + 132.96 P1AE0408 CD40 × FAP 2 + 1 head-to-tail  7.99 P1AA9663 CD40 × FAP2 + 2  3.65 P1AE0192 CD40 × FAP 1 + 1  1.15 P1AE0889 CD40 (VH2a/VF2a) ×FAP 24.26 4 + 1 with C-terminal crossfab P1AE0890 CD40 (VH2a/VF2a) × FAP30.42 4 + 1 with C-terminal crossfab

1.4.2 Binding to Human CD40-Expressing Daudi Cells

The binding to cell surface CD40 was tested using Daudi cells, a human Blymphoblast cell line with high expression levels of human CD40 (ATCCCCL-213). Daudi cells were cultured with 1× Dulbecco's Modified Eagle'sMedium (DMEM) (gibco, Cat. No. 42430-025) supplemented with 10% FetalBovine Serum (FBS) (life technologies, Cat. No. 16140, Lot No.1797306A). 0.3×10⁵ Daudi cells were added in 200 μl of 1× DMEM with 10%FBS to each well of a round-bottom 96-well plate (greiner bio-one,cellstar, Cat. No. 650185). Plates were centrifuged 5 minutes at 1700rpm and supernatants were flicked off. Cells were washed once with 200μL of 4° C. cold FACS buffer (eBioscience, Cat. No. 00-4222-26). Allsamples were resuspended in 50 μL/well of 4° C. cold FACS buffercontaining the bispecific antigen binding molecules (primary antibody)or isotype control antibody DP47 at the indicated range ofconcentrations (in duplicates) and incubated for 120 minutes at 4 ° C.Afterwards the cells were washed three times with 200 μL 4° C. cold FACSbuffer. Cells were further stained with 25 μL/well of 4° C. coldsecondary antibody solution (1:50 dilution of secondary antibody)containing R-Phycoerythrin (PE) conjugated AffiniPure F(ab′)₂ FragmentGoat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, Cat.No. 109-116-098) and incubated for 60 minutes at 4° C. in the dark.Cells were washed with 200 μl FACS buffer and resuspended in 85 μL/wellFACS-buffer containing 0.2 μg/mL DAPI (Roche, Cat. No. 10236276001) andacquired the same day using a 5-laser LSR-Fortessa (BD Bioscience withDIVA software). Data analysis was performed using the FlowJo version 10software (FlowJo LLC).

As shown in FIG. 18, all depicted clones bind to CD40 but vary in theirbinding strength (EC₅₀ values as well as signal strength) toCD40-positive Daudi cells. Bivalent anti-CD40 antibodies show higherEC₅₀ levels and reach higher binding plateaus compared to tetravalentanti-CD40 antibodies explained by more occupied CD40 binding sites perantibody and a gain of avidity of the tetravalent relative to thebivalent CD40 formats. The highest EC₅₀ value combined with the lowestbinding plateau was observed for the 1+1 format (P1AE0192). No bindingof the negative control antibody to Daudi cells was detected. The EC₅₀values as measured for different bispecific antibodies are shown inTable 8 below.

TABLE 8 Human CD40 binding characterization of CD40 antibodies indifferent bispecific antibody formats Molecule EC₅₀ [nM] P1AD4470 CD40IgG1 PGLALA 0.104 P1AE0637 CD40 × FAP 4 + 1 with C- 0.015 terminalcrossfab P1AD4453 CD40 × FAP 4 + 1 0.027 P1AD4574 CD40 × DP47 4 + 10.031 P1AD4455 CD40 × FAP 4 + 2 0.030 P1AD4465 CD40 × DP47 4 + 2 0.036P1AA9641 CD40 × FAP 2 + 1 0.079 P1AE0408 CD40 × FAP 2 + 1 head-to-tail0.044 P1AA9663 CD40 × FAP 2 + 2 0.096 P1AE0192 CD40 × FAP 1 + 1 21.628P1AE0889 CD40 (VH2a/VF2a) × FAP 0.046 4 + 1 with C-terminal crossfabP1AE0890 CD40 (VH2a/VL2a) × FAP 0.048 4 + 1 with C-terminal crossfab

Example 2 Functional Properties of FAP-Targeted Anti-Human CD40 BindingMolecules 2.1 CD40-Mediated Activation of Antigen Presenting Cells(APCs) by FAP-Targeted Anti-Human CD40 Binding Molecules

Ligation of CD40 induces B cell and dendritic cell (DC) maturation aswell as activation and promotes survival of these cell types. Upon CD40signaling cytokine production and costimulatory molecule expression onthe surface of B cells and DCs is increased (S. Quezada et al., AnnuRevImmunol. 2004, 22, 307-328; S. Danese et al., Gut. 2004, 53,1035-1043; G. Bishop et al., Adv Exp Med Biol. 2007, 597, 131-151).

In order to test the agonistic properties and the FAP specificity of thedifferent FAP-dependent anti-CD40 antibodies, APCs obtained from humanbuffy coats were incubated with the FAP-dependent agonistic anti-humanCD40 antibodies and either FAP-coated beads or human FAP expressingNIH/3T3 cells. APC activation was measured by FACS and supernatant ofthe cells was analyzed for cytokines by enzyme-linked immunosorbentassay (ELISA).

2.1.1 Activation of Human B Cells by FAP-Targeted Anti-Human CD40Binding Molecules using NIH/3T3-huFAP Cells as Source of Antigen

NIH/3T3 cells expressing FAP were used as source of antigen for thebispecific antigen binding molecules. The NIH/3T3-huFAP cells weregenerated by transfection of mouse embryonic fibroblast NIH/3T3 cells(ATCC CRL-1658) with the expression pETR4921 plasmid encoding human FAPunder a CMV promoter. The NIH/3T3-huFAP cells or NIH/3T3-wt cells wereirradiated with 27 Gy using a RS 2000 irradiator (Rad SourceTechnologies to prevent the cells from proliferating. 0.2×10⁵ irradiatedNIH/3T3 cells in 100 μl of R10 medium consisting of Roswell ParkMemorial Institute medium (RPMI) 1640 (gibco, Cat. No. 31870-025)supplied with 10% FBS, 1% (v/v) Penicillin Streptomycin (gibco, Cat. No.15070-063), 1% (v/v) L-Glutamine (gibco, Cat. No.25030-024), 1% (v/v)Sodium-Pyruvate (gibco, Cat. No. 11360-039), 1% (v/v) MEM non-essentialamino acids (gibco, Cat. No. 11140-035) and 50 μM (β-Mercaptoethanol(gibco, Cat. No. 31350-010) were seeded per well of a 96-wellflat-bottom plate (TPP, Cat. No. 92696).

On the next day a buffy coat was obtained from the Stiftung ZiircherBlutspendedienst SRK. In order to isolate peripheral blood mononuclearcells (PBMCs), 50 mL of buffy coat were diluted in the same volume ofPBS (gibco, Cat. No. 10010023). 50 mL polypropylene centrifuge tubes(TPP, Cat. No. 91050) were supplied with 15 mL of Lymphoprep™ (STEMCELLTechnologies, Cat. No. 07851) and 25 mL of the buffy coat/PBS solutionper tube were carefully layered above the Lymphorep™. The tubes werecentrifuged at 2000 rpm for 24 minutes at room temperature with lowacceleration and without break. Afterwards the PBMCs were collected fromthe interface, washed three times with PBS, resuspended in 10 mL of PBSand cells were analyzed for cell type and number with a Beckman Coultercell counter Ac·T™ 5diff OV (Beckman Coulter, Cat. No. 6605580). Priorto the B cell isolation from the PBMCs, the CD14-positive fraction wasremoved by magnetic labeling of the CD14-positive cells with CD14microbeads (Miltenyi, Cat. No. 130-050-201) and subsequent isolationwith the autoMACS® Pro Separator (Miltenyi, Cat. No. 130-092-545). TheCD14-negative fraction was used for subsequent B cell isolation with theMiltenyi B cell isolation kit II (Cat. No. 130-091-151) and autoMACS®separation. 1×10⁵B cells were added in 50 μl of R10 medium per well tothe NIH/3T3 cells. FAP-targeted anti-human CD40 antibodies were added in50 μl of R10 medium to the B cells at concentrations ranging from 1μg/mL to 0.3 ng/mL (3× dilution series). As positive control, theFAP-independent agonistic anti-human CD40 antibodies RO7009789 (IgG2,INN: Selicrelumab) and SGN-40 (IgG1, INN: Dacetuzumab) were used. Bothantibodies are bivalent for CD40. Since it is described in theliterature that the SGN-40 antibody requires Fc receptor cross-linkingfor biological activity (C. Law et al., Cancer Res 2005, 65, 8331-8338),a mechanism that is as well under discussion for RO7009789 (R. Dahan etal., Cancer Cell 2016, 29, 1-12), these two antibodies were incubatedwith a cross-linking goat anti-human IgG Fcγ fragment specific F(ab′)₂fragment (Jackson ImmunoResearch, Cat. No. 109-006-008) for 30 minutesbefore addition to the B cells. After 48 hours cells were transferredinto a 96-well round-bottom plate, washed once with PBS and incubatedwith 50 μl of 3 μg/mL of Fc receptor blocking Mouse IgG Isotype Control(ThermoFisher Scientific, Cat. No.10400C) in PBS. After 15 minutes ofincubation at 4° C., cells were washed with PBS and 50 μl of a mixtureof fluorescently labelled antibodies in PBS was added to the cells. Thefollowing fluorescently labelled antibodies were used: anti-human CD83BV421 (Biolegend, clone HB15e, Cat. No. 305324), anti-human CD80 BV605(BD Biosciences, clone L307.4, Cat. No. 563315), anti-HLA-ABC FITC (BDBiosciences, clone G46-2.6, Cat. No. 555552), anti-human CD14PerCP-Cy5.5 (Biolegend, clone HCD14, Cat. No. 325622), anti-human CD3PerCP-Cy5.5 (Biolegend, clone UCHT1, Cat. No. 300430), anti-human CD70PE (Biolegend, clone 113-16, Cat. No. 355104), anti-human CD86 PE-CF594(BD Biosciences, clone FUN-1, Cat. No. 562390), anti-HLA-DR APC (BDBiosciences, clone G46-6, Cat. No. 559866) and anti-human CD19 APC-H7(BD Biosciences, clone SJ25C1, Cat. No. 560177). In order to distinguishbetween live and dead cells, the viability dye Zombie Aqua™ (Biolegend,Cat. No. 423102) was added to the antibody mixture. After 30 minutes ofincubation at 4° C., cells were washed twice with PBS and thenresuspended in 200 μl of PBS. Cells were analyzed the same day using a5-laser LSR-Fortessa (BD Bioscience with DIVA software). Data analysiswas performed using the FlowJo version 10 software (FlowJo LLC). Live(aqua negative) cells, negative for CD14 and CD3 and positive for CD19were analyzed for CD70, CD80, CD83 and CD86 expression.

FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F, FIG. 3G, and FIG.3H show the FAP-dependent upregulation of B cell activation markers CD70(FIG. 3A and FIG. 3B), CD80 (FIG. 3C and FIG. 3D), CD83 (FIG. 3E andFIG. 3F) and CD86 (FIG. 3G and FIG. 3H) by bispecific antigen bindingmolecules tetravalent for human CD40 and either mono- or bivalent forFAP. The bispecific antibody monovalent for FAP induced a similarincrease of activation marker expression as the molecule with two FAPbinding moieties. With NIH/3T3-FAP cells upregulation of the B cellactivation markers by the bispecific antigen binding molecules wascomparable to the upregulation induced by the FAP-independent positivecontrol antibodies. Without FAP present (NIH/3T3-wt cells) no increaseof B cell activation markers was observed with the bispecific antigenbinding molecules, while positive control antibodies induced anupregulation in the expression of these markers.

2.1.2 Activation of Human Daudi Cells by FAP-Targeted Anti-Human CD40Binding Molecules using FAP-Coated Dynabeads® as Source of Antigen

1×10⁵ Daudi cells in 100 μl of 1× DMEM plus 10% FBS were added per wellof a 96-well flat-bottom plate. Instead of using cells expressing FAP assource of antigen, streptavidin Dynabeads® (ThermoFisher Scientific,Cat. No.: 11205D) were coated with biotinylated mouse FAP (producedin-house) (binding capacity of 6.5×10⁴ beads: 0.01 μg of protein)according to the manufacturer's instructions and added to the Daudicells in a bead:cell ratio of 2:1 in 50 μl of R10 medium. Usage of beadscoated with FAP instead of FAP-expressing cells provides a more stableand reproducible system, since fluctuating quality of the cells andsecretion of cellular products that might influence APC activationstatus represent factors that potentially distort results. As controlnon-coated beads were added to the Daudi cells. FAP-targeted anti-humanCD40 antibodies or positive control antibodies (described in section2.1.1) were added in 50 μl of R10 medium to the Daudi cells. After 2days Daudi cells were analyzed by FACS following the staining andanalysis procedures specified in 2.1.1.

B cells analyzed after 2 days of incubation with agonistic anti-CD40antibodies showed an increase in CD70 expression for all antibodies (seeFIG. 20A and FIG. 20B). The EC₅₀ values of specific molecules aresummarized in Table 9 below. Upregulation of these expression markerswas dependent on FAP in case of the different FAP-targeted antibodiesand increase of expression induced by these FAP-dependent antibodies washigher compared to the increase induced by the cross-linked CD40antibody (P1AD4470). In the absence of FAP (uncoated beads) no increaseof CD70 was observed with the depicted bispecific antibodies mono- orbivalent for CD40, while tetravalent CD40 binding molecules induced anupregulation of CD70, but to a lesser extent than in the presence of FAPindicating a low but detectable FAP-independent CD40 activation oftetravalent CD40 binder in Daudi cells.

TABLE 9 Activation of human Daudi cells using FAP-coated Dynabeads ®Molecule EC₅₀ [nM] P1AD4470 CD40 IgG1 0.756 P1AE0637 CD40 × FAP 4 + 1with C- 0.029 terminal crossfab P1AD4453 CD40 × FAP 4 + 1 0.015 P1AD4574CD40 × DP47 4 + 1 0.166 P1AD4455 CD40 × FAP 4 + 2 0.039 P1AD4465 CD40 ×DP47 4 + 2 n/a P1AA9641 CD40 × FAP 2 + 1 0.068 P1AE0408 CD40 × FAP 2 + 1head-to-tail 0.094 P1AA9663 CD40 × FAP 2 + 2 0.124 P1AE0192 CD40 × FAP1 + 1 0.409 P1AE0889 CD40 (VH2a/VF2a) × FAP 0.055 4 + 1 with C-terminalcrossfab P1AE0890 CD40 (VH2a/VF2a) × FAP 0.058 4 + 1 with C-terminalcrossfab

2.1.3 Activation of Human B Cells by FAP-Targeted Anti-Human CD40Binding Molecules using FAP-Coated Dynabeads® as Dource of Antigen

B cells were isolated from buffy coats as described in section 2.1.1 and1×10⁵B cells in 100 μl of R10 medium were added per well of a 96-wellflat-bottom plate. Streptavidin Dynabeads® (ThermoFisher Scientific,Cat. No.:11205D) were coated with biotinylated human or mouse FAP(produced in-house) (binding capacity of 6.5×10⁴ beads: 0.01 μg ofprotein) according to the manufacturer's instructions and added to the Bcells in a bead:cell ratio of 2:1 in 50 μl of R10 medium. As controlnon-coated beads were added to the B cells. The FAP-targeted anti-humanCD40 antibodies or positive control antibodies (described in section2.1.1) were added in 50 μl of R10 medium to the B cells. After 2 days Bcells were analyzed by FACS following the staining and analysisprocedures specified in 2.1.1. Alternatively B cells were cultured forfive days in presence of the agonistic anti-CD40 antibodies and 110 μlsupernatant were taken for IL-6 measurement using the human IL-6 DuoSetELISA kit (R&D, Cat. No. DY206-05). It is known that B cells produceincreased amounts of IL-6 upon stimulation with the CD40 ligand withoutthe need of additional B cell receptor stimuli (M. Duddy et al., J.Immunol. 2004, 172, 3422-3427). The ELISA was performed as described inthe protocol provided by the manufacturer with the only difference ofusing half of the recommended amounts for every step of the assay. Bcells were analyzed by FACS following the staining and analysisprocedures specified in section 2.1.1.

B cells analyzed after 2 days of incubation with agonistic anti-CD40antibodies showed an increase in CD70, CD83 and CD86 expression for allantibodies (see FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F,FIG. 21A and FIG. 21B and FIG. 25A, FIG. 25B, FIG. 25C, FIG. 25D). TheEC₅₀ values relating to the increase of CD86 expression of specificmolecules are summarized in Table 10 below. Upregulation of theseexpression markers was dependent on FAP in case of the differentFAP-targeted antibodies and increase of expression induced by theseFAP-dependent antibodies was comparable or slightly lower to theincrease induced by the cross-linked CD40 antibody P1AD4470.

TABLE 10 Activation of human B cells using FAP-coated Dynabeads ® shownas increase of CD86 expression Molecule EC₅₀ [nM] P1AD4470 CD40 IgG10.072 P1AE0637 CD40 × FAP 4 + 1 with C- 0.202 terminal crossfab P1AD4453CD40 × FAP 4 + 1 0.280 P1AD4574 CD40 × DP47 4 + 1 n/a P1AD4455 CD40 ×FAP 4 + 2 0.325 P1AD4465 CD40 × DP47 4 + 2 n/a P1AA9641 CD40 × FAP 2 + 10.909 P1AE0408 CD40 × FAP 2 + 1 head-to-tail 0.658 P1AA9663 CD40 × FAP2 + 2 1.004 P1AE0192 CD40 × FAP 1 + 1 0.742 P1AE0889 CD40 (VH2a/VF2a) ×FAP 0.329 4 + 1 with C-terminal crossfab P1AE0890 CD40 (VH2a/VF2a) × FAP0.345 4 + 1 with C-terminal crossfab

After 5 days of B cell incubation with FAP-coated Dynabeads®, antigenbinding molecules targeting human CD40 and FAP induced a FAP-dependentupregulation of CD80 expression on B cells. The levels of CD80expression induced by anti-human CD40 antibodies with either one or twoFAP binding sites were comparable. Treatment of B cells with positivecontrol anti-CD40 antibodies led to a similar extent of CD80upregulation. Elevated, FAP-dependent CD86 expression could be as welldetected with the bispecific antigen binding molecules. Again, presenceof one or two FAP binding sites made no major difference in expressionlevels of this activation marker. Compared to the FAP-independentupregulation of CD86 induced by cross-linked SGN-40 (dacetuzumab) oragonistic CD40 antibody selicrelumab (RO 7009789), CD86 upregulationinduced by FAP-dependent bispecific antigen binding molecules wasslightly lower. For CD70 and CD83 no or only very limited upregulationwas observed with the bispecifc antibodies targeting FAP and CD40, whilethe positive control antibodies clearly showed an effect on these B cellactivation markers (FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG.5F, FIG. 5G, FIG. 5H).

FIG. 6A and FIG. 6B show the effects of different agonistic anti-CD40antibodies on B cell IL-6 production. With FAP present IL-6concentration in the supernatants were elevated to a similar extent forall agonistic anti-human CD40 antibodies tested (about 6-fold increasecompared to untreated (UT) conditions). Upon incubation of the B cellswith non-coated Dynabeads® no increase in IL-6 production was detectedwith the bispecific antigen binding molecules, demonstratingFAP-dependency of these molecules.

2.1.4 Activation of Human Monocyte-Derived DCs (moDCs) by FAP-TargetedAanti-Human CD40 Binding Molecules using Human FAP-Coated Dynabeads® asSource of Antigen

PBMCs were isolated from buffy coats by Lymphoprep™ densitycentrifugation. Subsequently monocytes were isolated with CD14microbeads and autoMACS® separation as described in 2.1.1. In order togenerate monocyte-derived DCs (moDCs), 3×10⁶ monocytes were seeded perwell of a 6 well plate (TPP, Cat. No. 92006) with a density of 1×10⁶cells per mL. For DC maturation moDC medium consisting of RPMI 1640supplemented with 1% (v/v) Penicillin Streptomycin, 2% (v/v) human serum(heat inactivated for 30 minutes at 56° C.) (Sigma-Aldrich, Cat. No.H4522, Lot. No. SLBP1687V), 20 ng/mL freshly added recombinant humangranulocyte macrophage colony-stimulating factor (GM-CSF) (Peprotech,Cat. No. 300-03-20UG, Lot No. 081230H1213) and 20 ng/mL of freshly addedrecombinant human IL-4 (Peprotech, Cat. No. 200-04-100UG, Lot. No.09151403116) was used. After five days moDCs were harvested from the6-well plates by gentle removal of suspension and semi-adherent cellsand resuspended in fresh moDC medium containing human IL-4 and GM-CSF.2×10⁵ moDCs were seeded in 100 μl of moDC medium per well of a 96-wellflat-bottom plate. Streptavidin Dynabeads® were coated with biotinylatedhuman FAP and added in 50 μl in a bead/DC ratio of 2:1 (as described in2.1.3). As control non-coated beads were added to the moDCs.FAP-targeted anti-human CD40 antibodies were added in 50 μl to the DCsat concentrations ranging from 1 μg/mL to 0.3 ng/mL (3× dilutionseries). As positive, FAP-independent controls the agonistic anti-humanCD40 antibody RO7009789 and the cross-linked CD40 antibody were used.Two days after addition of the Dynabeads® and of the different agonisticanti-human CD40 antibodies moDC activation was measured by FACS. FACSstaining was performed as specified in 2.1.1 using a mixture offluorescently labelled antibodies consisting of anti-human CD86 BV421(BD Biosciences, clone FUN-1, Cat. No. 562432), anti-human CD80 BV605(BD Biosciences, clone L307.4, Cat. No. 563315), anti-HLA-ABC FITC (BDBiosciences, clone G46-2.6, Cat. No. 555552), anti-human CD1cPerCp-Cy5.5 (BD Biosciences, clone F10/21A3, Cat. No. 565424),anti-human CD70 PE (Biolegend, clone 113-16, Cat. No. 355104),anti-human CD11c PE-eF610 (eBioscience, clone 3.9, Cat. No. 61-0116-42),anti-human CD83 PE-Cy7 (BD Biosciences, clone HB15e, Cat. No. 561132),anti-human CD209 APC (BD Biosciences, clone DCN46, Cat. No. 551545),anti-human CD3 Alexa Fluor 700 (eBioscience, clone OKT3, Cat. No.56-0037-42), anti-human CD14 APC-H7 (BD Biosciences, clone M5E2, Cat.No. 561384) and viability dye Zombie Aqua™ (Biolegend, Cat. No. 423102).Cells were analyzed the same day using a 5-laser LSR-Fortessa. Dataanalysis was performed using the FlowJo version 10 software. Single,live cells were gated for CD3 negative and CD14 negative cells. Based onthis population CD lc and CD11c positive cells were analyzed for theexpression of the activation markers CD70, CD80, CD83 and CD86.

For all agonistic anti-human CD40 antibodies a pronounced and similarupregulation of CD83 on moDCs could be observed (FIG. 7A, FIG. 7B, FIG.7C, FIG. 7D, FIG. 7E, FIG. 7F). In case of the FAP-dependent anti-CD40antibodies, this upregulation was detected in a FAP-dependent manner.With the bispecifc antigen binding molecules targeting CD40 and FAP,CD80 expression was only slightly increased, however this increase wasFAP-dependent and comparable to the CD80 upregulation induced bypositive control antibody RO7009789 on the DCs (FIG. 7C and FIG. 7D).While CD86 expression was not significantly changed on DCs incubatedwith the different anti-CD40 antibodies compared to untreated DCs (FIG.7G and FIG. 7H), CD70 expression was elevated due to the agonisticeffects of these antibodies (FIG. 7A and FIG. 7B). Again bothFAP-dependent antibodies only showed activating properties when FAP waspresent and their effects on CD70 were similar. However numbers of moDCswith upregulated CD70 expression was in a low range (maximum 8% of CD11cand CD1c positive cells). RO7009789 showed a higher potency toupregulate CD70 on DCs compared to the bispecific molecules in thisexperimental setting.

2.1.5 Activation of HEK-Blue™ CD40L Cells by FAP-Targeted Anti-HumanCD40 Binding Molecules using Murine FAP-Coated Dynabeads® as Source ofAntigen

HEK-Blue™ CD40L cells (InvivoGen, Cat.No. hkb-cd40) were used as areporter cell line to analyze human CD40 stimulation mediated byFAP-targeted anti-human CD40 binding molecules. HEK-Blue™ CD40L cellsstably express the human CD40 receptor and NFκB-inducible secretion ofembryonic alkaline phosphatase (SEAP). Binding of CD40L or agonisticanti-CD40 antibody to the CD40 receptor expressed on HEK-Blue™ CD40Lcells triggers a signaling cascade leading to NFKB-mediated SEAPproduction. The amount of produced SEAP directly correlates with theextent of CD40 receptor activation. The levels of secreted SEAP in thesupernatant can be measured with a spectrophotometer at 620-655 nm.0.5×10⁵ HEK-Blue™ CD40L cells in 160 μl pre-warmed HEK-Blue™ detectionmedium (InvivoGen, Cat. No. hb-det2) were seeded per well of a 96-wellflat-bottom plate (TPP, Cat. No. 92696). Streptavidin Dynabeads® werecoated with biotinylated murine FAP and added in 20 μl PBS in abead:cell ratio of 2:1 (as described in 2.1.3). As control non-coatedbeads were added to the reporter cells. FAP-targeted anti-human CD40antibodies were added in 20 μl PBS to the cells at concentrationsranging from 6.89 nM to 0.0032 nM (3× dilution series). As controlmolecules antibodies tetravalent for human CD40 with one or two DP47domains instead of a FAP binding domain and a FAP-independentcross-linked CD40 antibody (P1AD4470) were used. After 8 hoursincubation at 37° C. SEAP levels in the supernatant were measured by aspectrophotometer at 650 nm.

For all agonistic FAP-targeted anti-human CD40 antibodies a pronouncedand comparable SEAP production was observed. In the case of FAP-targetedbispecific antibodies mono- or bivalent for human CD40 SEAP productionwas detected only in the presence of FAP. In contrast, reporter cellstreated with FAP-targeted bispecific antibodies tetravalent for humanCD40 secreted SEAP independently of FAP availability. However, in thepresence of FAP higher SEAP levels were detected in supernatant ofreporter cells treated with these antibodies. Moreover, the negativecontrol antibodies tetravalent for human CD40 with one or two DP47domains instead of a FAP binding domain induced comparable SEAPproduction in HEK-Blue™ CD40L cells in the presence and absence of FAP.The positive control antibody CD40 IgG (P1AD4470) +F(ab) induced similarlevels of SEAP production as compared to FAP-targeted bispecificantibodies bivalent or tetravalent for human CD40 in the presence ofFAP-coated beads (FIG. 8A, FIG. 8B, FIG. 19A and FIG. 19B). The EC₅₀values as measured for the CD40 antibody as well as for differentbispecific antibodies are shown in Table 11 below.

TABLE 11 Activation of HEK-Blue ™ CD40L cells using murine FAP-coatedDynabeads ® Molecule EC₅₀ [nM] P1AD4470 CD40 IgG1 PGLALA 0.125 P1AE0637CD40 × FAP 4 + 1 with C- 0.031 terminal crossfab P1AD4453 CD40 × FAP 4 +1 0.067 P1AD4574 CD40 × DP47 4 + 1 0.421 P1AD4455 CD40 × FAP 4 + 2 0.077P1AA9641 CD40 × FAP 2 + 1 0.197 P1AE0408 CD40 × FAP 2 + 1 head-to-tail0.167 P1AA9663 CD40 × FAP 2 + 2 0.238 P1AE0192 CD40 × FAP 1 + 1 0.639P1AE0889 CD40 (VH2a/VF2a) × FAP 0.044 4 + 1 with C-terminal crossfabP1AE0890 CD40 (VH2a/VF2a) × FAP 0.0005 4 + 1 with C-terminal crossfab

2.2 CD40-Mediated Activation of DCs by FAP-Targeted Anti-CD40 BindingMolecules and Subsequent Priming of T Cells

In order to demonstrate the ability of DCs activated by theFAP-dependent anti-human CD40 antibodies to efficiently prime T cells,in vitro T cell priming assays were established. For these assays DCsfrom the spleens of transgenic mice expressing the human CD40 receptor(huCD40tg mice) (mice with similar human and murine CD40 receptorexpression pattern; C57BL/6 background; generated by Taconic) wereisolated, pulsed with either SIINFEKL peptide or with ovalbumin (OVA)(DEC-205 receptor-mediated antigen uptake) and incubated with differentagonistic anti-human CD40 antibodies. FAP was provided via FAP-coatedDynabeads® in order to show FAP-dependency of the bispecific antigenbinding molecules. 24 hours later CD8 positive T cells were isolatedfrom spleens of OT1 mice (CD8-positive T cells of these mice all possessa transgenic TCR recognizing SIINFEKL in the context of H2-Kb;C57BL/6-Tg(TcraTcrb)1100Mjb/Crl, Charles River), carboxyfluoresceinsuccinimidyl ester (CFSE) labelled and added to the pulsed DCs. On dayfive of the experiment s DC and T cell cytokines were analyzed in thesupernatants of the cultured cells and T cells were analyzed foractivation and proliferative capabilities.

2.2.1 T Cell Priming via SIINFEKL-Pulsed DCs Activated by FAP-TargetedAnti-CD40 Binding Molecules

DCs were isolated from the spleens of huCD40tg mice. In order to isolatesplenic DCs, the spleen from a huCD40tg mouse was put into one well of a6-well plate containing 2.25 mL Hank's Balanced Salt Solution (HBSS)with Calcium²+(gibco, Cat. No. 14025-05), 250 μl of a 10 mg/mL solutionof collagenase D (end concentration 1 mg/mL) (Sigma-Aldrich, Cat. No.11088866001) and 12.5 μl of a 10 mg/mL DNase solution (end concentration0.05 mg/mL) (Sigma-Aldrich, D5025-150KU, Lot. No. SLBRO535V). Using a 3mL syringe (BD, Cat. No. 309658) with a 21G needle (Braun, Cat. No.4657527) the spleen was ballooned and subsequently, with the help ofscissors, torn into small pieces. After a 25 minutes incubation at 37°C., 50 μL of 0.5 M ethylenediaminetetraacetic acid (EDTA) (Applichem,Cat. No. A4892.1000) were added, followed by a second incubation step at37° C. for five minutes. The solution containing splenocytes and smallpieces of splenic tissue was filtered through a 40 μm filter (Corning,Cat. No. 352340) into a 50 mL polypropylene centrifuge tube. Splenictissue pieces were smashed through the filter with the end of a 3 mLsyringe plug. In the next step the 50 mL tube was centrifuged at 1500rpm for 5 minutes at room temperature, the supernatant was discarded and1 mL of 1× cell lysis buffer (diluted 1:10 with distilled water) (BD,Cat. No. 555899) was added to the splenocytes in order to lyse the redblood cells. After four minutes of incubation at room temperature, 20 mLof R10 were added followed by a centrifugation step at 1500 rpm for 5minutes at room temperature. The supernatant was removed, thesplenocytes were resuspended in 30 mL of R10 and cell count as well asviability were determined with the automated EVE cell counter (VWR, Cat.No. 734-2675). The mouse CD11 c UltraPure microbeads (Miltenyi, Cat. No.130-108-338) were used according to the manufacturer's instruction toisolate DCs by autoMACS® separation. Subsequently 0.25×10⁵ DCs wereseeded in 50 μl of R10 per well of a 96-well flat-bottom plate. The DCswere then pulsed with SIINFEKL peptide (Ovalbumin residues 257-264)(Eurogentec, Cat. No. AS-60193-5, Lot. No. 1360618) in a suboptimalconcentration of 1 pg/mL. This limited amount of antigen allowsdetecting variances in T cell activation due to differently activatedDCs. As a positive control in order to induce high T cell activationindependent of additional DC activating stimuli, SIINFEKL was added at aconcentration of 1 ng/mL to the DCs. DCs that were not pulsed with theSIINFEKL antigen served as negative control. Human FAP-coated ornon-coated Dynabeads® were added in 50 μL of R10 to the DCs at a 2:1bead:cell ratio. In the next step different agonistic anti-CD40antibodies were added in 50 μL of R10 at concentrations ranging from 1μg/mL to 1 ng/mL (10× dilution series). In this experimental setup, thebispecific anti-human CD40 antibody containing two FAP binding sites wascompared to its equivalent with two DP47 domains instead of FAP bindingdomains, to the FAP-independent RO7009789 and cross-linked SGN-40 and toa murine FAP-dependent bispecific antibody tetravalent for murine CD40and bivalent for FAP (28H1 FAP binder). After 24 hours splenicCD8-positive cells from OT1 mice were isolated. In order to do so, thespleen of an OT1 mouse was smashed through a 40 μm filter with the endof a 3 mL syringe plug into a 50 mL tube. The filter was washed with R10and the splenocytes were centrifuged at 1500 rpm for 5 minutes at roomtemperature. 1 mL of 1× cell lysis buffer (diluted 1:10 with distilledwater) was added to the cells and after four minutes of incubation atroom temperature, 20 mL of R10 were added. The tube was centrifuged at1500 rpm for 5 minutes at room temperature and the supernatant wasdiscarded. The splenocytes were resuspended in 30 mL of R10 and cellcounts as well as viability were determined with the automated EVE cellcounter. CD8-positive cells were isolated in a negative selectionprocess using the mouse CD8a±T Cell Isolation Kit (Miltenyi, Cat. No.130-104-075) and autoMACS® separation according to the manufacturer'sinstructions. CD8-positive cells that were found in the negativefraction after the separation were then washed with pre-warmed PBS,counted with the EVE cell counter and the cell number was adjusted to2×10⁷ cells/mL in pre-warmed PBS. 10 mM CFSE solution (CellTrace™ CFSECell Proliferation Kit, ThermoFisher, Cat. No. C34554) was 5000-folddiluted in pre-warmed PBS and added to the cells resuspended in PBS in a1:1 ratio (CFSE end concentration 1 μM). After a short vortex, cellswere incubated for five minutes at room temperature. The labellingreaction was stopped by adding 40 mL of pre-warmed R10 medium to thecells. After two washing steps with PBS, CD8-positive cells wereresuspended in R10 and 0.5×10⁵ cells were added in 100 μl R10 to thepulsed DCs. On day five of the experiment T cells were restimulated forintracellular cytokine staining (ICS) with 0.5 μg/mL of SIINFEKL and 2μg/mL anti-mouse CD28 antibody (eBioscience, clone 37.51, Cat. No.16-0281-86). One hour after SIINFEKL and anti-CD28 addition, Brefeldin A(BFA) (BD, Cat. No. 51-2301KZ) (1:1000) was added to the cells in orderto block intracellular protein transport. After another four hoursincubation step, 150 μl of the supernatant were taken for Luminex™ basedmultiplexed cytokine measurement. In order to measure a set of 23cytokines, the Bio-Plex Pro™ Mouse Cytokine GrpI Panel 23-Plex kit(BioRad, Cat. No. M60009RDPD) was used according to the manufacturer'sinstructions. For flow cytometry analysis of the T cells, cells in the96-well flat-bottom plates were transferred into 96-well round-bottomplates, washed once with PBS and incubated with 50 μl of 3 μg/mL of Fcreceptor blocking Mouse IgG Isotype Control in PBS. After 15 minutes ofincubation at 4° C., cells were washed with PBS and 50 μl of a mixtureof fluorescently labelled antibodies in PBS was added to the cells. Thefollowing antibodies were used: anti-mouse CD86 BV785 (Biolegend, cloneGL-1, Cat. No. 105043), anti-I-A/I-E PerCp-Cy5.5 (Biolegend, cloneM5/114.15.2, Cat. No. 107626), anti-mouse CD70 PE (eBioscience, cloneFR70, Cat. No. 12-0701-82), anti-mouse CD3 PE-CF594 (BD Biosciences,clone 145-2C11, Cat. No. 562286), anti-mouse CD25 PE-Cy7 (eBioscience,clone PC61.5, Cat. No. 25-0251-82), anti-mouse CD11c APC (BDBiosciences, clone HL3, Cat. No. 561119), anti-mouse CD44 Alexa Fluor700 (BD Biosciences, clone IM7, Cat. No. 560567) and anti-mouse CD8APC-Cy7 (Biolegend, clone 53-6.7, Cat. No. 100714). In order todistinguish between live and dead cells, the viability dye Zombie Aqua™was added to the antibody mixture. Cells were incubated for 30 minutesat 4° C. with the extracellular staining antibody solution. Afterwardscells were washed two times with PBS, permeabilized and intracellularlystained for IFNγ using anti-mouse IFNγ BV421 (Biolegend, clone XMG1.2,Cat. No. 505830) with the Foxp3/Transcription Factor Staining Buffer Set(eBioscience, Cat. No. 00-5523-00) according to the manufacturer'sprotocol. Cells were resuspended in 200 μl of PBS and analyzed the sameday using a 5-laser LSR-Fortessa. Data analysis was performed using theFlowJo version 10 software. The population of live cells that displayedexpression of CD8 and CD3 was analyzed for CFSE dye dilution, IFNγproduction, CD44 and CD25 expression.

FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, FIG. 9F, FIG. 9G, and FIG.9H show that DCs pulsed with low amounts of SIINFEKL and stimulated withdifferent agonistic anti-CD40 antibodies are able to induce T cellproliferation. In case of the FAP-dependent bispecific anti-CD40antibodies proliferation is only induced when FAP is provided in theassay. Levels of proliferation induced by DCs stimulated with the murineor the human version of the bispecific antigen binding molecules withfour CD40 and two FAP binding moieties was comparable. This stronglysuggests that downstream signaling of the human CD40 receptor expressedon DCs is not impaired in the huCD40tg mice. No significant upregulationof the T cell activation markers CD44 and CD25 or IFNγ production wasobserved for T cells cocultured with DCs that have been stimulated withdifferent agonistic anti-CD40 antibodies. Only DCs pulsed with highamounts of SIINFEKL displayed clear changes of these markers compared tothe untreated condition.

Cytokine concentration measurement in the supernatant showed an effectof the agonistic anti-CD40 antibodies on IL-2 (FIG. 9I) and IL-12(p40)(FIG. 9J) expression. With the FAP-dependent human anti-CD40 antibodyelevated IL-12(p40) levels were detected only in the presence of FAP.However, the murine equivalent bispecific antigen binding moleculeinduced a markedly higher secretion of IL-12(p40). IL-2 secretion wasincreased to a similar extent with both, the anti-human CD40 and theanti-mouse CD40 bispecific antigen binding molecules in a FAP-dependentway.

2.2.2 T Cell Priming Via OVA-Pulsed DCs Activated by FAP-TargetedAnti-CD40 Binding Molecules

DCs were isolated from the spleens of huCD40tg mice and FAP-coated ornon-coated Dynabeads® as well as different agonistic anti-CD40antibodies were added to the splenic DCs as described in section 2.2.1.Instead of pulsing DCs with SIINFEKL, which requires no uptake andprocessing by the DCs, OVA protein was used as antigen. In order topromote the OVA uptake in a Toll-like receptor (TLR) stimulusindependent way (additional TLR stimuli might lead to a high overallactivation of DCs, making the detection of different activation statesdue to stimulation with agonistic anti-CD40 antibodies impossible) theOva Antigen Delivery Reagent (Miltenyi, Cat. No. 130-094-663) incombination with a biotinylated anti-mouse DEC205 antibody (Miltenyi,clone NLDC-145, Cat. No. 130-101-854) was used according to themanufacturer's protocol. In brief, DCs are incubated with a biotinylatedantibody that binds to the DEC205 receptor, which is highly expressed onCD8-positive cross-presenting DCs (M. Lahoud et al., Int Immunol. 2000,12(5), 731-735). Afterwards the Ova delivery reagent, an anti-biotinantibody coupled to FITC and OVA, is added to the cells leading toDEC205 receptor-mediated uptake of OVA. In order to provide a negativecontrol, DCs were only labelled with the anti-DEC205 antibody withoutthe addition of OVA. On the next day, CD8-positive T cells were isolatedfrom OT1 mice, CFSE labelled and added to the DCs as described insection 2.2.1. On day five of the experiment 150 μl of supernatant weretaken for IFNγ measurement using the Mouse IFN-gamma DuoSet ELISA kit(R&D, Cat. No. DY485-05). The ELISA was performed as described in theprotocol provided by the manufacturer. For FACS analysis of the T cells,cells in the 96-well flat-bottom plates were transferred into 96-wellround-bottom plates, washed once with PBS and incubated with 50 μl of 3μg/mL of Fc receptor blocking Mouse IgG Isotype Control in PBS. After 15minutes of incubation at 4° C. cells were washed with PBS and 50 μl of amixture of fluorescently labelled antibodies in PBS were added to thecells. The following antibodies were used: anti-mouse CD4 BV421(Biolegend, clone GK1.5, Cat. No. 100438), anti-mouse CD86 BV785(Biolegend, clone GL-1, Cat. No. 105043), anti-I-A/I-E PerCp-Cy5.5

(Biolegend, clone M5/114.15.2, Cat. No. 107626), anti-mouse CD70 PE(eBioscience, clone FR70, Cat. No. 12-0701-82), anti-mouse CD3 PE-CF594(BD Biosciences, clone 145-2C11, Cat. No. 562286), anti-mouse CD25PE-Cy7 (eBioscience, clone PC61.5, Cat. No. 25-0251-82), anti-mouseCD11c APC (BD Biosciences, clone HL3, Cat. No. 561119), anti-mouse CD44Alexa Fluor 700 (BD Biosciences, clone IM7, Cat. No. 560567) andanti-mouse CD8 APC-Cy7 (Biolegend, clone 53-6.7, Cat. No. 100714). Inorder to distinguish between live and dead cells, the viability dyeZombie Aqua™ was added to the antibody mixture. Cells were incubated for30 minutes at 4° C. with 50 μl of the staining antibody mix. Afterwardscells were washed two times with PBS, resuspended in 200 μl of PBS andanalyzed using 5-laser LSR-Fortessa. Data analysis was performed usingthe FlowJo version 10 software. Viable CD3- and CD8-positive cells wereanalyzed for CFSE signal, CD25 and CD44 expression.

FIG. 10A, FIG. 10B, FIG. 10C, FIG. 11A, FIG. 11B, FIG. 11C, FIG. 12A,FIG. 12B, FIG. 12C and FIG. 22A, and FIG. 22B show that DCs incubatedwith the OVA delivery reagent and stimulated with the bispecific antigenbinding molecule targeting human CD40 and FAP can significantly enhanceCD8 positive OT1 T cell proliferation (FIG. 10A, FIG. 10B, FIG. 10C andFIG. 22A, FIG. 22B) as well as expression of the T cell activationmarkers CD25 (FIG. 11A, FIG. 11B, and FIG. 11C) and CD44 (FIG. 12A, FIG.12B, and FIG. 12C). These effects were FAP-dependent. The results of theIFNγ ELISA confirm the enhanced T cell activation due to the humananti-CD40 FAP-targeted antibody: IFNγ levels were elevated in conditionswith T cells cocultured with DCs treated with the anti-human CD40 FAPtargeting antibody (FIG. 13A, FIG. 13B, FIG. 13C). Effects of the murineanti-CD40 FAP-targeted antibody were comparable, underpinning thehypothesis that the huCD40tg mouse model provides a suitable system formeasuring the effects of agonistic anti-human CD40 antibodies.

Example 3 Functional Properties of FAP-Targeted Anti-Murine CD40 BindingMolecules 3.1 CD40-Mediated In Vitro Activation of Murine B Cells byFAP-Targeted Anti-Murine CD40 Binding Molecules

Spleens from C57BL/6J mice were processed as described in section 2.2.1.Murine B cells were isolated from splenocytes using the mouse B cellisolation kit (Miltenyi, Cat. No. 130-090-862) according to themanufacturer's instructions. 1×10⁵ B cells were seeded in 100 μl R10 perwell of a 96-well flat-bottom plate. Dynabeads® coated with murinebiotinylated FAP (in-house production) (see 2.1.3 for detaileddescription) or non-coated Dynabeads as control were added in 50 μl R10in a bead:cell ratio of 2:1. Agonistic anti-murine CD40 antibodies wereadded in 50 μl of R10 to the B cells. Antibody concentrations variedfrom 1 μg/mL to 0.3 ng/mL (3× dilution series). In this experimentalsetup bispecific antigen binding molecules carrying four anti-mouse CD40binding sites and either one or two FAP binding sites (28H1 FAP binder,equivalent to the FAP binding domain in anti-human CD40 bispecificantigen binding molecules) were compared to the FAP-independent FGK4.5antibody (rat IgG2a, BioXcell Catalogue No. BE0016-2), which is bivalentfor murine CD40. The biological activity of FGK4.5 is dependent on Fcreceptor cross-linking (L. Richman et al., Cancer Immunol Res. 2014,2(1)), therefore FGK4.5 was pre-incubated for 30 minutes at roomtemperature with a goat anti-rat IgG (H+L) cross-linking antibody(Jackson ImmunoResearch, Cat. No. 112-005-003, Lot. No. 123801). After48 hours B cells were analyzed for expression of activation markers byFACS. For this purpose, B cells were transferred into 96-wellflat-bottom plates, washed with PBS and incubated with 50 μl of 3 μg/mLof Fc receptor blocking Mouse IgG Isotype Control in PBS. After 15minutes of incubation at 4° C., cells were washed with PBS and 50 μl ofa mixture of fluorescently labelled antibodies in PBS was added to thecells. The following antibodies were used: anti-mouse CD19 BV605 (BDBiosciences, clone 1D3, Cat. No. 563148), anti-mouse CD86 BV785(Biolegend, clone GL-1, Cat. No. 105043), anti-I-A/I-E PerCp-Cy5.5(Biolegend, clone M5/114.15.2, Cat. No. 107626), anti-mouse CD70 PE(eBioscience, clone FR70, Cat. No. 12-0701-82), anti-mouse CD80 PE-CF594(BD Biosciences, clone 16-10A1, Cat. No. 562504), anti-mouse CD3 PE-Cy7(BD Biosciences, clone 145-2C11, Cat. No. 552774), anti-mouse NK1.1PE-Cy7 (Biolegend, clone PK136, Cat. No. 108714), anti-mouse CD11c APC(BD Biosciences, clone HL3, Cat. No. 561119), anti-mouse CD45 AlexaFluor 700 (eBioscience, clone 30-F11, Cat. No. 56-0451-82), anti-mouseCD8 APC-Cy7 (Biolegend, clone 53-6.7, Cat. No. 100714). To distinguishbetween live and dead cells, the viability dye Zombie Aqua™ was added tothe antibody mixture. Cells were incubated for 30 minutes at 4° C. withthe staining mixture, washed two times with PBS and then resuspended in200 μl of PBS. FACS analysis was performed with a 5-laser LSR-Fortessaand data analysis was conducted using the FlowJo version 10 software.Viable, single cells were gated for CD11c-negative, CD3-negative andNK1.1-negative cells in order to exclude non-B cells. CD8-negative,CD19-positive cells were analyzed for expression of the B cellactivation markers CD70, CD80, CD83 and CD86.

Incubation of murine B cells with the FAP-targeted anti-mouse CD40antibodies with either one or two FAP binding moieties increasedexpression of B cell activation markers. CD70 expression was onlyslightly increased with the bispecific antigen binding moleculescompared to conditions with the cross-linked FGK4.5 antibody (FIG. 14Aand FIG. 14B). However, overall numbers of cells expressing high levelsof CD70 were rather low. CD80 expression was upregulated in aFAP-dependent manner upon treatment of B cells with the bispecificmolecules targeting murine CD40 and FAP (FIG. 14C and FIG. 14D).Bispecific molecules showed higher potency than the bivalentFAP-independent FGK4.5 antibody in case of CD80 upregulation. CD86expression increase was as well induced by all agonistic anti-CD40antibodies tested and expression levels were comparable for allantibodies (FIG. 14E and FIG. 14F). While CD86 upregulation wasFAP-dependent with the tetravalent anti-mouse CD40 antibody possessingtwo FAP binding moieties, a FAP-independent effect was observed for thebispecific antigen binding molecule having only one FAP binding site. Inaddition, a FAP-independent upregulation of MHC-II expression wasobserved for all tested bispecific antigen binding molecules (FIG. 14Gand FIG. 14H)

3.2 CD40-Mediated In Vivo Activation of Murine DCs and T Cells byFAP-Targeted Anti-Murine CD40 Binding Molecules

The murine colon adenocarcinoma MC38_FAP transfectant tumor cell linewith mouse FAP expression was obtained from an in-vivo-passage performedat Roche Glycart AG and after expansion deposited in the Glycartinternal cell bank. MC38_muFAP_invipa cells were cultured in DMEMcontaining 10% FCS (PAA Laboratories, Austria), 1 mM Pyruvate, 1× NEAAand 6 μg/ml Puromycine. Cells were cultured at 37° C. in awater-saturated atmosphere at 5% CO2 and were injected at in vitropassage 14 at a viability of 95%. 2×10⁶ tumor cells were injectedsubcutaneously in a 100 μl cell suspension (50% RPMI medium and 50%matrigel). 33 C57B1/6 female mice with an age of 8-9 weeks at start ofthe experiment (purchased from Charles Rivers, Germany) were maintainedunder specific-pathogen-free condition with daily cycles of 12 h lightand 12 h darkness according to committed guidelines (GV-Solas; Felasa;TierschG). Experimental study protocol was reviewed and approved bylocal government (P 2011-128) and after arrival animals were maintainedfor one week to get accustomed to the new environment and forobservation. They were afterwards implanted with a transpondersubcutaneously on the right side of the back for identification andmaintained one more week for recovery. Continuous health monitoring wascarried out on regular basis. To study the FAP-targeted activation ofCD40 in vivo mice were injected subcutaneously on study day 0 with 2×10⁶of MC38-FAP. At day x when tumors reached 200 mm³, 9 mice per group wereinjected i.p. with 200 μL of the different compounds. Mice in thevehicle group were injected with Histidine buffer and animals in thetreatment groups were either injected with 10 mg/kg FGK4.5 or 15 mg/kgFGK4.5 4+1. Animals were controlled daily for clinical symptoms anddetection of adverse effects such as weight loss. Termination criteriafor animals were clinical sickness, impaired locomotion and scruffy fur.At the time points 72 h and 8 d post therapy injection, tumor, spleen,tumor-draining and tumor-non-draining lymph nodes were collected fromthree mice per group and analyzed by flow cytometry. In addition, serumfrom all sacrificed animals was collected to analyze serum enzymesindicative of liver injury.

For flow cytometer analysis single cell suspensions of all collectedorgans were prepared and stained with fluorescently labelled antibodiesas described in section 2.1.1. and section 3.1, respectively. Todistinguish between live and dead cells, the viability dye Zombie Aqua™was added to the antibody mixture. Cells were incubated for 30 minutesat 4° C. with the staining mixture, washed two times with PBS andresuspended in 200 μl of PBS. FACS analysis was performed with a 5-laserLSR-Fortessa and data analysis was conducted using the FlowJo version 10software. DCs were identified as viable, single cells highly positivefor CD11c and MHC class II and negative for CD3, NK1.1 and CD19. CD70and CD86 expression, both DC activation markers, was analyzed on DCsthree days post therapy injection. Viable CD45-, CD3- and CD8-positivesingle cells were identified as CD8⁺ T cells. CD8⁺ T cells were analyzedfor Ki67 FITC (eBioscience, clone SolA15, Cat. No. 11-5698-82)expression and total numbers of CD8⁺ T cell in the tumors weredetermined using absolute cell count beads (Invitrogen, Cat. No.01-1234).

As shown in FIG. 23A, FIG. 23B, and FIG. 23C, on day 3 enzymessuggestive of hepatocellular injury were increased in mice injected witha single i.p. dose of CD40 but not in mice injected FAP-CD40 or vehiclealone. In addition, the body weight was deceased and the spleen weightwas increased of CD40-treated mice three days post treatment compared tomice injected with FAP-CD40 4+1 or vehicle alone (FIG. 23D and FIG. 23E,respectively) indicating less severe side effects in animals treatedwith the FAP-targeted CD40 antibodies compared to animals treated withthe parental CD40 antibody. Although to a lesser extent than FGK4.5,FAP-targeted anti-CD40 antibodies induced a significant increase in DCactivation (CD86 and CD70 expression) in tumor-draining lymph nodesthree days post treatment (FIG. 24A and FIG. 24B) and CD8⁺ T cellproliferation (Ki68 expression and cell numbers) in tumors eight dayspost treatment (FIG. 24C and FIG. 24D) compared to vehicle-treated mice.In summary, the FAP-targeted anti-CD40 molecule with FAP-dependentactivation of CD40 in a 4+1 format induces potent DC and T cellactivation in tumor-bearing mice with reduced systemic toxicity comparedto the untargeted anti-CD40 parental antibody FGK4.5.

Example 4 Generation and Production of Humanized Variants of Anti-CD40Antibody S2C6 4.1 First Generation of Humanized Variants of Anti-CD40Antibody S2C6 4.1.1 Methodology

Anti-CD40 antibody S2C6 is disclosed in WO 2000/075348 and has the VHdomain of SEQ ID NO:129 and the VL domain of SEQ ID NO:130. Variantsthereof were created as described in the following. For theidentification of a suitable human acceptor framework during thehumanization of the anti-CD40 binder S2C6, a combination of twomethodologies was used. On the one hand, a classical approach was takenby searching for an acceptor framework with high sequence homology,grafting of the CDRs on this framework, and evaluating whichback-mutations can be envisaged. More explicitly, each amino aciddifference of the identified frameworks to the parental antibody wasjudged for impact on the structural integrity of the binder, and backmutations towards the parental sequence were introduced wheneverappropriate. The structural assessment was based on Fv region homologymodels of both the parental antibody and its humanized versions createdwith an in-house antibody structure homology modeling tool implementedusing the Biovia Discovery Studio Environment, version 4.5.

On the other hand, an in-house developed in silico tool was used topredict the orientation of the VH and VL domains of the humanizedversions towards each other (see WO 2016062734 incorporated herein byreference). The results were compared to the predicted VH-VL domainorientation of the parental binder to select for framework combinationswhich are close in geometry to the starting antibody. The rational is todetect possible amino acid exchange in the VH-VL interface region thatmight lead to disruptive changes in the pairing of the two domains.

4.1.2 Choice of Acceptor Framework and Adaptations Thereof

The acceptor framework was chosen as described in Table 12 below:

TABLE 12 Acceptor framework Choice of human Identity to human V- MurineV-region acceptor V-region region germline after germline germlinegrafting (BLASTp): S2C6 VH IGHV1-26*01 IGHV1-2*01 91.8% S2C6 VLIGKV1-110*01 IGKV2-30*02 92.0%

Post-CDR3 framework regions were adapted from human IGHJ germlineIGHJ6*01/02 (YYYYYGMDVWGOGTTVTVSS) and human IGKJ germline IGKJ1*01(WTFGOGTKVEIK). The part relevant for the acceptor framework isindicated as underlined.

Based on structural considerations, back mutations from the humanacceptor framework to the amino acid in the parental binder wereintroduced at positions H48 (M>I) and H71 (R>V) of the VH region and atpositions L36 (F>Y), L46 (R>L) and L87 (Y>F) of the VL region (Kabatnumbering). Furthermore, two positions in CDR-H2 were identified aspromising candidates for forward mutations, i.e., amino acid exchangesfrom parental binder to human acceptor germline in order to increaseoverall human character, namely H60 (N>A) and H64 (K>Q).

In order to address putative developability hotspots (asparaginedeamidation), further changes with regard to the parental binder wereintroduced at positions H52b (N>Q) and H54 (N>A) in VH, and L27f (N>Q),L28 (G>P), L29 (N>Q) and L30 (T>I) in VL (Kabat numbering).

In the following Table 13 the VH-VL pairing matrix is shown:

hVK_6 hVK_9 hVK_1 hVK_4 hVK_5 bF36Y_(—) hVK_7 hVK_8 bF36Y_(—) IMGT_(—)hVK_3 bF36Y_(—) bF36Y_(—) bR46L_(—) bF36Y_(—) bF36Y_(—) bR46L_(—)hVK_2_(—) hVK_2 bF36Y_(—) bR46L_(—) bR46L_(—) bY87F_(—) bR46L_(—)bR46L_(—) bY87F_(—) 30_base_(—) bF36Y_(—) bR46L_(—) bY87F_(—) bY87F_(—)dG28P_(—) bY87F_(—) bY87F_(—) dN27fQ_(—) graft bR46L bY87F dG28P dT30IdT30I dN27fQ dN29Q dN29Q hVH_1 IMGT_hVH_1_(—) 2_base_graft hVH_2bM48I_bR71V x x x x x x x x hVH_3 bM48I_bR71V_(—) x x x x x x x x dN54AhVH_4 bM48I_bR71V_(—) x x x x x x x x dN52bQ_dN254A hVH_5bM48I_bR71V_(—) x x fK64Q hVH_6 bM48I_bR71V_(—) x x fN60A hVH_7bM48I_bR71V_(—) x x fN60A_fK64QBack mutations prefixed with b, forward mutations prefixed with f, andmutations to address developability hotspots prefixed with d

4.1.3 VH and VL domains of the resulting humanized CD40 antibodies

The resulting VH domains of humanized CD40 antibodies can be found inTable 14 below and the resulting VL domains of humanized CD40 antibodiesare listed in Table 15 below.

TABLE 14 Amino acid sequences of the VH domains ofhumanized CD40 antibodies Seq ID Description Sequence No IMGT_hVH_1QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 45GLEWMGrvipnnggtsynqkfkgRVTSTRDTSISTAYMELSRL RSDDTVVYYCARegiywWGQGTTVTVSSIMGT_hVH_2 QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 46GLEWIGrvipnnggtsynqkfkgRVTSTVDTSISTAYMELSRL RSDDTVVYYCARegiywWGQGTTVTVSSIMGT_hVH_3 QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 47GLEWIGrvipnaggtsynqkfkgRVTSTVDTSISTAYMELSRL RSDDTVVYYCARegiywWGQGTTVTVSSIMGT_hVH_4 QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 48GLEWIGrvipqaggtsynqkfkgRVTSTVDTSISTAYMELSRL RSDDTVVYYCARegiywWGQGTTVTVSSIMGT_hVH_5 QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 49GLEWIGrvipnnggtsynqkfqgRVTSTVDTSISTAYMELSRL RSDDTVVYYCARegiywWGQGTTVTVSSIMGT_hVH_6 QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 50GLEWIGrvipnnggtsyaqkfkgRVTSTVDTSISTAYMELSRL RSDDTVVYYCARegiywWGQGTTVTVSSIMGT_hVH_7 QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 51GLEWIGrvipnnggtsyaqkfqgRVTSTVDTSISTAYMELSRL RSDDTVVYYCARegiywWGQGTTVTVSSIMGT_hVH_2_N288A QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 52GLEWIGrvipnaggtsynqkfkgRVTSTVDTSISTAYMELSRL RSDDTVVYYCARegiywWGQGTTVTVSSIMGT_hVH_5_N288A QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 53GLEWIGrvipnaggtsynqkfqgRVTSTVDTSISTAYMELSRL RSDDTVVYYCARegiywWGQGTTVTVSSIMGT_hVH_6_N288A QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 54GLEWIGrvipnaggtsyaqkfkgRVTSTVDTSISTAYMELSRL RSDDTVVYYCARegiywWGQGTTVTVSSIMGT_hVH_7_N288A QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 55GLEWIGrvipnaggtsyaqkfqgRVTSTVDTSISTAYMELSRL RSDDTVVYYCARegiywWGQGTTVTVSS

TABLE 15 Amino acid sequences of the VL domains ofhumanized CD40 antibodies Seq ID Description Sequence No IMGT_hVK_1DVVMTQSPLSLPVTLGQPASISCrssqslvhsngntflhWFQQ 56RPGQSPRRLIYtvsnrfsGVPDRFSGSGSGTDFTLKISRVEAE DVGVYYCsqtthvpwtFGQGTKVEIKIMGT_hVK_2 DVVMTQSPLSLPVTLGQPASISCrssqslvhsngntflhWYQQ 57RPGQSPRLLIYtvsnrfsGVPDRFSGSGSGTDFTLKISRVEAE DVGVYYCsqtthvpwtFGQGTKVEIKIMGT_hVK_3 DVVMTQSPLSLPVTLGQPASISCrssqslvhsngntflhWYQQ 58RPGQSPRLLIYtvsnrfsGVPDRFSGSGSGTDFTLKISRVEAE DVGVYFCsqtthvpwtFGQGTKVEIKIMGT_hVK_4 DVVMTQSPLSLPVTLGQPASISCrssqslvhsnpntflhWYQQ 59RPGQSPRLLIYtvsnrfsGVPDRFSGSGSGTDFTLKISRVEAE DVGVYFCsqtthvpwtFGQGTKVEIKIMGT_hVK_5 DVVMTQSPLSLPVTLGQPASISCrssqslvhsngniflhWYQQ 60RPGQSPRLLIYtvsnrfsGVPDRFSGSGSGTDFTLKISRVEAE DVGVYFCsqtthvpwtFGQGTKVEIKIMGT_hVK_6 DVVMTQSPLSLPVTLGQPASISCrssqslvhsnpniflhWYQQ 61RPGQSPRLLIYtvsnrfsGVPDRFSGSGSGTDFTLKISRVEAE DVGVYFCsqtthvpwtFGQGTKVEIKIMGT_hVK_7 DVVMTQSPLSLPVTLGQPASISCrssqslvhsqgntflhWYQQ 62RPGQSPRLLIYtvsnrfsGVPDRFSGSGSGTDFTLKISRVEAE DVGVYFCsqtthvpwtFGQGTKVEIKIMGT_hVK_8 DVVMTQSPLSLPVTLGQPASISCrssqslvhsngqtflhWYQQ 63RPGQSPRLLIYtvsnrfsGVPDRFSGSGSGTDFTLKISRVEAE DVGVYFCsqtthvpwtFGQGTKVEIKIMGT_hVK_9 DVVMTQSPLSLPVTLGQPASISCrssqslvhsqgqtflhWYQQ 64RPGQSPRLLIYtvsnrfsGVPDRFSGSGSGTDFTLKISRVEAE DVGVYFCsqtthvpwtFGQGTKVEIK

The humanized amino acid sequences for heavy and light chain variableregions of S2C6 variants were backtranslated in to DNA and the resultingcNDA were synthesized (GenArt) and then cloned into heavy chainexpression vectors as fusion proteins with human IgG1 backbones/humanCH1-Hinge-CH2-CH3 with LALA and PG mutations (Leucine 234 to Alanine,Leucine 235 to Alanine, Proline 329 to Glycine) abrogating effectorfunctions or into light chain expression vectors as fusion proteins tohuman C-kappa. LC and HC Plasmids were then cotransfected into HEK293and purified after 7 days from supernatants by standard methods forantibody purification.

4.2 Second Generation of Humanized Variants of Anti-CD40 Antibody S2C64.2.1 Methodology

As for Example 4.1, for the identification of a suitable human acceptorframework during the humanization of the anti-CD40 binder S2C6 acombination of two methodologies was used. On the one hand, a classicalapproach was taken by searching for an acceptor framework with highsequence homology, grafting of the CDRs on this framework, andevaluating which back-mutations can be envisaged. More explicitly, eachamino acid difference of the identified frameworks to the parentalantibody was judged for impact on the structural integrity of thebinder, and back mutations towards the parental sequence were introducedwhenever appropriate. The structural assessment was based on Fv regionhomology models of both the parental antibody and its humanized versionscreated with an in-house antibody structure homology modeling toolimplemented using the Biovia Discovery Studio Environment, version 4.5.

On the other hand, an in-house developed in silico tool was used topredict the orientation of the VH and VL domains of the humanizedversions towards each other (see WO 2016062734 incorporated herein byreference). The results were compared to the predicted VH-VL domainorientation of the parental binder to select for framework combinationswhich are close in geometry to the starting antibody. The rational is todetect possible amino acid exchange in the VH-VL interface region thatmight lead to disruptive changes in the pairing of the two domains.

4.2.2 Choice of Acceptor Framework and Adaptations Thereof

Two different acceptor frameworks were chosen as described in Table 16and Table 18 below.

TABLE 16 Acceptor framework 1: “IGHV1-IGKV2D” Choice of human Identityto human V- Murine V-region acceptor V-region region germline aftergermline germline grafting (BLASTp): S2C6 VH IGHV1-26*01 IGHV1-2*0591.8% S2C6 VL IGKV1-110*01 IGKV2D-29*02 88.0%

Post-CDR3 framework regions were adapted from human IGHJ germlineIGHJ6*01/02 (YYYYYGMDVWGQGTTVTVSS) and human IGKJ germline IGKJ4*01/02(LTFGGGTKVEIK). The part relevant for the acceptor framework isindicated in bold script.

Based on structural considerations, back mutations from the humanacceptor framework to the amino acid in the parental binder wereintroduced at positions H43 (Q>K), H44 (G>S), H69 (M>L), H71 (R>V), H73(T>K), H88 (V>A) and H105 (Q>H) of the VH region and at positions L2(I>V), L4 (M>V), L87 (Y>F) and L104 (V>L) of the VL region. In onevariant, mutation T70S (VH) was included to study the effect of aslightly more hydrophilic residue at this position.

All variants include the N54A mutation (VH) to address a putativedevelopability hotspot (asparagine deamidation). All positions are givenin the Kabat EU numbering scheme.

In the following Table 17 the Humanization variant VH-VL pairing matrixis shown:

VL1d VL1c bI2V, VL1b bI2V, bM4V, VL1a bM4V, bM4V, bY783F, bY87F bY87FbY83F bV104L VH1a bG44S, bM69L, bR71V, bT73K, bV88A x x x x VH1b bQ43K,bG44S, bM69L, bR71V, bT73K, x x x x bV88A VH1c bG44S, bM69L, bR71V,bT73K, bV88A, x x x x bQ105H VH1d bG44S, bM69L, bR71V, bT73K, bV88A, x xx x xT70SMutation N54A applies to all VH variants and is not explicitlymentioned. Back mutations prefixed with b, forward mutations prefixedwith f, and other mutations prefixed with x

TABLE 18 Acceptor framework 2: “IGHV3-IGKV1” Choice of human Identity tohuman V- Murine V-region acceptor V-region region germline aftergermline germline grafting (BLASTp): S2C6 VH IGHV1-26*01 IGHV3-23*0279.6% S2C6 VL IGKV1-110*01 IGKV1-39*01 79.0%

Post-CDR3 framework regions were adapted from human IGHJ germlineIGHJ6*01/02 (YYYYYGMDVWGQGTTVTVSS) and human IGKJ germline IGKJ4*01/02(LTFGGGTKVEIK). The part relevant for the acceptor framework isindicated in bold script.

Based on structural considerations, back mutations from the humanacceptor framework to the amino acid in the parental binder wereintroduced at positions H44 (G>S), H49 (S>G), H71 (R>V), H78 (L>A), H94(K>R) and H105 (Q>H) of the VH region and at positions L42 (K>Q), L43(A>S) and L87 (Y>F) of the VL region. Furthermore, four positions inCDR-H2 were identified as promising candidates for forward mutations,i.e., amino acid exchanges from parental binder to human acceptorgermline in order to increase overall human character, namely H60 (N>G),H61 (Q>D), H62 (K>S) and H63 (F>V).

All variants include the N54A mutation (VH) to address a putativedevelopability hotspot (asparagine deamidation). All positions are givenin the Kabat EU numbering scheme.

In the following Table 19 the Humanization variant VH-VL pairing matrixis shown:

VL2b bK42Q, VL2a bA43S, bY87F bY87F VH2a bS49G, bR71V, bL78A, bK94R x xVH2b bG44S, bS49G, bR71V, bL78A, bK94R x x VH2c bS49G, bR71V, bL78A,bK94R, bQ105H x x VH2d bS49G, fN6OG, fQ61D, fK62S, fF63V, x x bR71V,bL78A, bK94R

Back mutations prefixed with b, forward mutations prefixed with f.

4.2.3 VH and VL Domains of the Resulting Humanized CD40 Antibodies

The resulting VH and VL domains of humanized CD40 antibodies based onacceptor framework 1 can be found in Table 17 below and the resulting VHand VL domains of humanized CD40 antibodies based on acceptor framework2 are listed in Table 18 below.

TABLE 20 Amino acid sequences of the VH and VL domains ofhumanized CD40 antibodies based on acceptor framework 1 Seq IDDescription Sequence No VH1a QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ171 SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGQGTTVTVSS VH1bQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGK 172SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL RSDDTAVYYCAREGIYWWGQGTTVTVSSVH1c QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 173SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL RSDDTAVYYCAREGIYWWGHGTTVTVSSVH1d QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 174SLEWMGRVIPNAGGTSYNQKFKGRVTLSVDKSISTAYMELSRL RSDDTAVYYCAREGIYWWGQGTTVTVSSVL1a DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 175KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE DVGVYFCSQTTHVPWTFGGGTKVEIKVL1b DIVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 176KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE DVGVYFCSQTTHVPWTFGGGTKVEIKVL1c DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 177KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE DVGVYFCSQTTHVPWTFGGGTKVEIKVL1d DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 178KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE DVGVYFCSQTTHVPWTFGGGTKLEIK

TABLE 21 Amino acid sequences of the VH and VL domains ofhumanized CD40 antibodies based on acceptor framework 2 Seq IDDescription Sequence No VH2a EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK179 GLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTTVTVSS VH2bEVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 180SLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSL RAEDTAVYYCAREGIYWWGQGTTVTVSSVH2c EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 181GLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSL RAEDTAVYYCAREGIYWWGHGTTVTVSSVH2d EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 182GLEWVGRVIPNAGGTSYGDSVKGRFTISVDNSKNTAYLQMNSL RAEDTAVYYCAREGIYWWGQGTTVTVSSVH2ab EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYMHWVRQAPGK 183GLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSL RAEDTAVYYCAREGIYWWGQGTTVTVSSVH2ac EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 184GLEWVGRVIPNAGGTSYNQKVKGRFTISVDNSKNTAYLQMNSL RAEDTAVYYCAREGIYWWGQGTTVTVSSVL2a DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 185KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE DFATYFCSQTTHVPWTFGGGTKVEIKVL2b DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 186KPGQSPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE DFATYFCSQTTHVPWTFGGGTKVEIKVL2ab DIQMTQSPSSLSASVGDRVTITCRASQSLVHSNGNTFLHWYQQ 187KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE DFATYFCSQTTHVPWTFGGGTKVEIKVL2ac DIQMTQSPSSLSASVGDRVTITCRSSQSIVHSNGNTFLHWYQQ 188KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE DFATYFCSQTTHVPWTFGGGTKVEIK

4.2.4 New Humanized CD40 Antibodies in huIgG1_LALA_PG Format

Based on the new humanization variants of VH and VL new CD40 antibodieswere expressed as huIgG1 antibodies with an effector silent Fc (P329G;L234, L235A) to abrogate binding to Fcγ receptors according to themethod described in WO 2012/130831 A1.

TABLE 22 Nomenclature for VH/VL combinations expressed as huIgG1_LALA_PGantibodies VL1a VL1b VL1c VL1d VL2a VL2b VL2ab VL2ac VH1a P1AE P1AE P1AEP1AE 0817 1001 0993 0996 VH1b P1AE P1AE P1AE P1AE 1002 1003 1004 1005VH1c P1AE P1AE P1AE P1AE 0997 1006 0818 0998 VH1d P1AE P1AE P1AE P1AE0999 1007 1000 0819 VH2a P1AE P1AE 0400 0404 VH2b P1AE P1AE 0401 0405VH2c P1AE P1AE 0402 0406 VH2d P1AE P1AE 0403 0407 VH2ab P1AE P1AE 11251126 VH2ac P1AE P1AE 1134 1135

The full-length sequences of humanized CD40 antibodies as humanIgG1_LALAPG antibodies can be found in Table 20.

TABLE 23 Amino acid sequences of the humanizedCD40 IgG1_LALAPG antibodies Seq ID Antibody Sequence No P1AE0400EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 189 heavy chainGLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0400DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 190 light chainKPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0401EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 191 heavy chainSLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0401DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 192 light chainKPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0402EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 193 heavy chainGLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGHGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0402DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 194 light chainKPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0403EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 195 heavy chainGLEWVGRVIPNAGGTSYGDSVKGRFTISVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0403DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 196 light chainKPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0404EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 197 heavy chainGLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0404DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 198 light chainKPGQSPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0405EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 199 heavy chainSLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0405DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 200 light chainKPGQSPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0406EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 201 heavy chainGLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGHGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0406DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 202 light chainKPGQSPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0407EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 203 heavy chainGLEWVGRVIPNAGGTSYGDSVKGRFTISVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0407DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 204 light chainKPGQSPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0816QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 259 heavy chainSLEWMGRVIPNNGGTSYNQKFQGRVTISVDKSISTAYMELSSL (control)RSEDTAVYYCAREGIYWWGHGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0816DVVVTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTFLHWYLQ 260 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE (control)DVGVYFCSQTTHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0817QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 205 heavy chainSLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0817DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 206 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0818QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 207 heavy chainSLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGHGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0818DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 208 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0819QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 209 heavy chainSLEWMGRVIPNAGGTSYNQKFKGRVTLSVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0819DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 210 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0993QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 211 heavy chainSLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0993DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 212 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0996QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 213 heavy chainSLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0996DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 214 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0997QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 215 heavy chainSLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGHGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0997DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 216 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0998QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 217 heavy chainSLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGHGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0998DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 218 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0999QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 219 heavy chainSLEWMGRVIPNAGGTSYNQKFKGRVTLSVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0999DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 220 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1000QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 221 heavy chainSLEWMGRVIPNAGGTSYNQKFKGRVTLSVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1000DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 222 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1001QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 223 heavy chainSLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1001DIVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 224 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1002QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGK 225 heavy chainSLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1002DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 226 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1003QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGK 227 heavy chainSLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1003DIVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 228 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1004QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGK 229 heavy chainSLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1004DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 230 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1005QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGK 231 heavy chainSLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1005DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 232 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1006QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 233 heavy chainSLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGHGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1006DIVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 234 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1007QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 235 heavy chainSLEWMGRVIPNAGGTSYNQKFKGRVTLSVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1007DIVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 236 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1125EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYMHWVRQAPGK 237 heavy chainGLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1125DIQMTQSPSSLSASVGDRVTITCRASQSLVHSNGNTFLHWYQQ 238 light chainKPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1126EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYMHWVRQAPGK 239 heavy chainGLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1126DIQMTQSPSSLSASVGDRVTITCRSSQSIVHSNGNTFLHWYQQ 240 light chainKPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1135EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 241 heavy chainGLEWVGRVIPNAGGTSYNQKVKGRFTISVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1135DIQMTQSPSSLSASVGDRVTITCRSSQSIVHSNGNTFLHWYQQ 242 light chainKPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

4.2.5 Production of the New Humanized CD40 Antibodies in huIgG1_LALA_PGFormat

The antibodies were expressed by transient transfection of HEK293-Fcells grown in suspension with expression vectors encoding the differentpeptide chains. Transfection into HEK293-F cells (Invitrogen, USA) wasperformed according to the cell supplier's instructions using Maxiprep(Qiagen, Germany) preparations of the antibody vectors, F17 based medium(Invitrogen, USA), PEIpro (Polyscience Europe GmbH) and an initial celldensity of 1-2 million viable cells/ml in serum free FreeStyle 293expression medium (Invitrogen). Cell culture supernatants were harvestedafter 7 days of cultivation in shake flasks or stirred fermenters bycentrifugation at 14000 g for 30 minutes and filtered through a 0.22 μmfilter.

The antibodies were purified from cell culture supernatants by affinitychromatography using MabSelectSure-Sepharose ™ (GE Healthcare, Sweden)chromatography. Briefly, sterile filtered cell culture supernatants werecaptured on a MabSelect SuRe resin equilibrated with PBS buffer (10 mMNa₂HPO_(4, 1) mM KH₂PO₄, 137 mM NaCl and 2.7 mM KCl, pH 7.4), washedwith equilibration buffer and eluted with 25 mM citrate, pH 3.0. Afterneutralization with 1 M Tris pH 9.0, aggregated protein was separatedfrom monomeric antibody species by size exclusion chromatography(Superdex 200, GE Healthcare) in 20 mM histidine, 140 mM NaCl, pH 6.0.Monomeric protein fractions were pooled, concentrated if required usinge.g. a MILLIPORE Amicon Ultra (30KD MWCO) centrifugal concentrator andstored at −80° C. Sample aliquots were used for subsequent analyticalcharacterization e.g. by CE-SDS, size exclusion chromatography, massspectrometry and endotoxin determination.

The production yield for the different humanized CD40 antibodies isshown in Table 21 as titer values calculated from the yield afterpreparative affinity chromatography using MabSelectSure-Sepharose™chromatography.

Purity and molecular weight of the molecule after the final purificationstep were analyzed by CE-SDS analyses in the presence and absence of areducing agent. The Caliper LabChip GXII system (Caliper Lifescience)was used according to the manufacturer's instruction.

The aggregate content of the molecule was analyzed using a TSKgel G3000SW XL analytical size-exclusion column (Tosoh) in 25 mM potassiumphosphate, 125 mM sodium chloride, 200 mM L-arginine monohydrocloride,0.02% (w/v) NaN₃, pH 6.7 running buffer at 25° C.

For direct comparison of all antibodies the thermal stability wasmonitored by Static Light Scattering (SLS) and by measuring theintrinsic protein fluorescence in response to applied temperaturestress. 30 μg of filtered protein sample with a protein concentration of1 mg/ml was applied in duplicate to an Optim 2 instrument (AvactaAnalytical Ltd). The temperature was ramped from 25 to 85° C. at 0.1°C./min, with the radius and total scattering intensity being collected.For determination of intrinsic protein fluorescence the sample wasexcited at 266 nm and emission was collected between 275 nm and 460 nm.For all antibodies the aggregation temperature (Tagg) was between 64 and69° C. and is provided in Table 24 or Table 25 below.

The production yield for the humanized CD40 antibodies with thedifferent frameworks is shown in Table 24 or Table 25 below.

TABLE 24 Production titer, humanness and aggregation temperature ofhumanized CD40 antibodies based on acceptor framework 2 Titer humannessAntibody VH/VL [μg/ml] (VH/VL in %) Tagg P1AD4470 control 140 77.6/78 68P1AE0400 VL2a/VH2a 219 77.6/78 69 P1AE0401 VL2a/VH2b 162 76.5/78 69P1AE0402 VL2a/VH2c 196 77.6/78 69 P1AE0403 VL2a/VH2d 137 80.6/78 67P1AE0404 VL2b/VH2a 165 77.6/76 69 P1AE0405 VL2b/VH2b 128 76.5/76 69P1AE0406 VL2b/VH2c 154 77.6/76 69 P1AE0407 VL2b/VH2d 102 80.6/76 67

TABLE 25 Production titer, humanness and aggregation temperature ofhumanized CD40 antibodies based on acceptor framework 1 Titer humannessAntibody VH/VL [μg/ml] (VH/VL in %) Tagg P1AE0816 control 8.5 84.7/84 64P1AE0817 VH1a/VL1a 62 86.7/87 67 P1AE0818 VH1c/VL1c 47 86.7/85 66P1AE0819 VH1d/VL1d 90 85.7/85 67 P1AE0993 VH1a/VL1c 34 86.7/85 67P1AE0996 VH1a/VL1d 16 86.7/85 67 P1AE0997 VH1c/VL1a 44 86.7/87 66P1AE0998 VH1c/VL1d 24 86.7/85 66 P1AE0999 VH1d/VL1a 34 85.7/87 67P1AE1000 VH1d/VL1c 16 85.7/85 66 P1AE1001 VH1a/VL1b 34 86.7/86 65P1AE1002 VH1b/VL1a 46 85.7/87 67 P1AE1003 VH1b/VL1b 49 85.7/86 66P1AE1004 VH1b/VL1c 60 85.7/85 67 P1AE1005 VH1b/VL1d 7 85.7/85 65P1AE1006 VH1c/VL1b 24 86.7/86 65 P1AE1007 VH1d/VL1b 34 85.7/86 67

4.2.6 Generation of Recombinant Human and Cynomolgus Monkey CD40Extracellular Domain Protein

Following constructs were cloned and expressed by transient expressionin HEK293 cells:

1) Human CD40 extracellular domain (amino acids 21-193 of SEQ ID NO:1,NCBI accession number NP_001241) with C-terminal His-AviTag™ tag (SEQ IDNO:266)

2) Cynomolgus monkey (macaca fascicularis) CD40 extracellular domain(amino acids 21-193, cynomolgus CD40 extracellular domain sequence wastaken from Roche cynomolgus cDNA database, unpublished data) withC-terminal His-AviTag™ tag (SEQ ID NO:267)

CD40 extracellular domain antigens for binding analysis were generatedby gene synthesis (Eurofins Genomics GmbH service, Germany), cloned viaunique restriction sites into Roche's in house expression vector usingstandard cloning procedures. Cloning of all constructs was verified bysequencing. All antigens were expressed under the control of theCMV-promoter. For transient expression of the CD40 extracellular domainconstructs, suspension-adapted HEK293-F cells (Life Technologies, USA)were transfected with the respective plasmids: In general, 1L ofHEK293-F cells at about 2×10⁶ cells/ml were transfected with a total of500 μg plasmid DNA complexed by the PEIpro Transfection Reagent(Polysciences Europe GmbH, Germany) according to manufacturer'sinstructions. After transfection, HEK293-F cells were incubated for 6days. The cells were subsequently harvested by centrifugation and theprotein-containing supernatant was filtered using a 0.22 μm vacuumfiltration system (Millipore). The His-AviTag™ tagged proteins werepurified by IMAC affinity chromatography using complete-His-Tag resin(Roche Diagnostics). After washing with 50 mM Na₂PO₄, 300 mM NaCl, pH8.0, His-AviTag™ fusion proteins were eluted using washing buffersupplemented with 500 mM Imidazol at pH 7.0. Aggregated protein wasseparated from monomeric fusion proteins by size exclusionchromatography (Superdex 75, GE Healthcare) in 20 mM Tris, 150 mM NaCl,pH 7.4. Monomeric protein fractions were pooled, concentrated ifrequired using e.g. a MILLIPORE Amicon Ultra (10KD MWCO) centrifugalconcentrator and stored at −80° C. Sample aliquots were used forsubsequent analytical characterization e.g. by CE-SDS, size exclusionchromatography and mass spectrometry.

Biotinylation of CD40 Extracellular Domain:

Enzymatic site specific biotinylation of human or cynomolgus CD40extracellular domain constructs containing a C-terminal AviTag™ wasperformed by using the BirA biotin-protein ligase kit (Avidity LLC, USA)according to manufactures instruction. Briefly, 1/10 volume of BiomixA(10× concentration: 0.5M bicine buffer, pH 8.3) and BiomixB (10×concentration: 100 mM ATP, 100 mM MgOAc, 500 μM d-biotin) was added toAviTag™ containing protein followed by addition of 2.5 μg BirA ligaseper 10 nmol protein. The reaction mixture was incubated at 30° C. for 1h and purified by size exclusion chromatography on a Superdex75 prepgrade prepacked HiLoad column (GE Healthcare, Sweden).

4.2.7 Human/Cynomolgus CD40 Binding Surface Plasmon ResonanceSpectroscopy Assay

Around 12000 resonance units (RU) of the capturing system (10 μg/ml goatanti human F(ab)′₂; Order Code: 28958325; GE Healthcare Bio-Sciences AB,Sweden) were coupled on a CM5 chip (GE Healthcare BR-1005-30) at pH 5.0by using an amine coupling kit supplied by the GE Healthcare. The sampleand system buffer was PBS-T (10 mM phosphate buffered saline including0.05% Tween20) pH 7.4. The flow cell was set to 25° C.—and the sampleblock set to 12° C.—and primed with running buffer twice. The antibodywas captured by injecting a 50 nM solution for 30 sec at a flow of 5μl/min. Association was measured by injection of human CD40 extracellular domain or cynomolgus monkey CD40 extracellular domain invarious concentrations in solution for 300 sec at a flow of 30 μl/minstarting with 300 nM in 1:3 dilutions. The dissociation phase wasmonitored for up to 1200 sec and triggered by switching from the samplesolution to running buffer. The surface was regenerated by 60 secwashing with a Glycine pH 2.1 solution at a flow rate of 30 μl/min. Bulkrefractive index differences were corrected by subtracting the responseobtained from a goat anti human F(ab′)₂ surface. Blank injections arealso subtracted (=double referencing). For calculation of apparent K_(D)and other kinetic parameters the Langmuir 1:1 model was used. Theapparent Kd was calculated using the Biacore™ B4000 evaluation software(version 1.1).

4.2.8 Cellular Binding Assay for Characterisation of CD40-SpecificHumanized Antibodies

CD40 positive cells (Raji cells) were detached from the culture bottleusing Trypsin and were counted using a Casy cell counter. Afterpelleting at 4° C., the cells were resuspended in FACS Buffer (2.5% FCSin PBS), adjusted to 2.0E+06 cells/mL, and dispensed to 96-well PPV-bottom-plates (25 μL/well=5.0E+04Zellen/well).

The CD40 specific antibodies were adjusted to 20 μg/mL in FACS buffer,resulting in a final concentration of 10 μg/mL. 20 μl were added to 25μl cell suspension and incubated for 1 h at 4° C. The cells were thenwashed twice in FACS buffer. After washing, the cells were resuspendedin 50 μL FACS-buffer containing secondary antibody (<huIgG>-Alexa488,c=10 μg/mL) and incubated 1 h bei 4° C. The cells were then washed twicein FACS buffer and resuspended in 70 μl/well FACS buffer for measurementusing a FACS Canto (BD, Pharmingen).

In Table 26 the affinity of the humanized CD40 antibodies (measured byBiacore) and the cellular binding to CD40 expressing cells (Raji cells)is shown.

TABLE 26 Affinity and cellular binding of humanized CD40 antibodies toCD40 expressing cells EC₅₀ [μg/ml] cellular Affinity binding ID VH/VL[nM] Ka (1/Ms) Kd (1/s) (Raji) P1AD4470 control 4.6 1.69E+06 7.81E−030.09 P1AE0400 VL2a/VH2a 4.2 1.68E+06 6.99E−03 0.12 P1AE0401 VL2a/VH2b4.6 1.69E+06 7.87E−03 0.13 P1AE0402 VL2a/VH2c 4.2 1.67E+06 7.09E−03 0.13P1AE0403 VL2a/VH2d 29 1.40E+06 4.07E−02 0.12 P1AE0404 VL2b/VH2a 4.21.63E+06 6.93E−03 0.11 P1AE0405 VL2b/VH2b 5.1 1.61E+06 8.14E−03 0.09P1AE0406 VL2b/VH2c 4.2 1.67E+06 7.09E−03 0.09 P1AE0407 VL2b/VH2d 301.19E+06 3.55E−02 0.12 P1AE0816 control 8.7 2.53E+06 2.19E−02 0.09P1AE0817 VH1a/VL1a 2.5 2.40E+06 5.93E−03 0.09 P1AE0818 VH1c/VL1c 3.22.63E+06 8.47E−03 0.14 P1AE0819 VH1d/VL1d 3.4 2.59E+06 8.77E−03 0.11P1AE0993 VH1a/VL1c 3.4 2.68E+06 8.98E−03 0.13 P1AE0996 VH1a/VL1d 3.52.59E+06 9.08E−03 0.12 P1AE0997 VH1c/VL1a 2.3 2.59E+06 6.03E−03 0.12P1AE0998 VH1c/VL1d 3.3 2.70E+06 8.96E−03 0.12 P1AE0999 VH1d/VL1a 2.42.45E+06 5.92E−03 0.15 P1AE1000 VH1d/VL1c 3.2 2.68E+06 8.62E−03 0.14P1AE1001 VH1a/VL1b 2.7 2.56E+06 6.81E−03 0.08 P1AE1002 VH1b/VL1a 2.22.54E+06 5.57E−03 0.13 P1AE1003 VH1b/VL1b 2.5 2.46E+06 6.06E−03 0.13P1AE1004 VH1b/VL1c 3 2.63E+06 7.95E−03 0.14 P1AE1005 VH1b/VL1d 3.22.58E+06 8.16E−03 0.11 P1AE1006 VH1c/VL1b 2.6 2.53E+06 6.51E−03 0.14P1AE1007 VH1d/VL1b 2.7 2.50E+06 6.62E−03 0.12

4.2.9 Antibody Characterisation by UHR-ESI-QTOF Mass Spectrometry

The samples were desalted by HPLC on a Sephadex G25 5×250 mm column(Amersham Biosciences, Freiburg, Germany) using 40% acetonitrile with 2%formic acid (v/v). The total mass was determined by UHR-ESI-QTOF MS on amaXis 4G UHR-QTOF MS system (Bruker Daltonik, Bremen, Germany) equippedwith a TriVersa NanoMate source (Advion, Ithaca, N.Y.). Data acquisitionwas done at 900-4000 m/z (ISCID: 0.0 eV). The raw mass spectra wereevaluated and transformed into individual relative molar masses using anin-house developed software tool.

4.2.10 Thermal Stability Evaluation of Antibodies

Samples are prepared at a concentration of 1 mg/mL in 20 mMHistidine/Histidine chloride, 140 mM NaCl, pH 6.0, transferred into anoptical 384-well plate by centrifugation through a 0.4 μm filter plateand covered with paraffine oil. The hydrodynamic radius is measuredrepeatedly by dynamic light scattering on a DynaPro Plate Reader (Wyatt)while the samples are heated with a rate of 0.05° C./min from 25 ° C. to80 ° C. Alternatively, samples were transferred into a 10 μLmicro-cuvette array and static light scattering data as well asfluorescence data upon excitation with a 266 nm laser were recorded withan Optim1000 instrument (Avacta Inc.), while they were heated at a rateof 0.1° C./min from 25° C. to 90° C. The aggregation onset temperatureis defined as the temperature at which the hydrodynamic radius (DLS) orthe scattered light intensity (Optim1000) starts to increase. Themelting temperature is defined as the inflection point in a graphshowing fluorescence intensity vs. wavelength.

Example 5 Generation and Production of Bispecific Constructs with NewHumanized CD40 Antibody Variants 5.1 Generation of Bispecific AntigenBinding Molecules Targeting CD40 and Fibroblast Activation Protein (FAP)

The cDNAs encoding a VH domain and a VL domain as described in Example 4were cloned in frame with the corresponding constant heavy or lightchains of human IgG1 in suitable expression plasmids. Expression ofheavy and light chain is driven by a chimeric MPSV promoter consistingof the MPSV core promoter and a CMV enhancer element. The expressioncassette also contains a synthetic polyA signal at the 3′ end of thecDNAs. In addition the plasmid vectors harbor an origin of replication(EBV OriP) for episomal maintenance of the plasmids.

In analogy to Example 1, different bispecific CD40-FAP antibodies areprepared in 4+1 format consisting of four CD40 binding moieties combinedwith one FAP binding moiety at the C-terminus of an Fc (FIG. 1A) or in2+1 and 2+2 formats consisting of two CD40 binding moieties combinedwith either one FAP binding moiety at the C-terminus of an Fc (FIG. 1Cand FIG. 1E) or two FAP binding moieties at the C-terminus of an Fc(FIG. 1D). In addition, a bispecific antibody consisting of one CD40binding moiety combined with one FAP binding moiety is prepared (FIG.1F). The generation and preparation of FAP binders 28H1 and 4B9 isdescribed in WO 2012/020006 A2, which is incorporated herein byreference. To generate the 4+1 and the 2+1 molecules the knob-into-holetechnology is used to achieve heterodimerization. The S354C/T366Wmutations are introduced in the first heavy chain HC1 (Fc knob heavychain) and the Y349C/T366S/L368A/Y407V mutations are introduced in thesecond heavy chain HC2 (Fc hole heavy chain). In the 2+2 molecule theCrossMab technology as described in WO 2010/145792 A1 ensures correctlight chain pairing. Independent of the bispecific format, in all casesan effector silent Fc (P329G; L234, 234A) is used to abrogate binding toFcγ receptors according to the method described in WO 2012/130831 A1.Sequences of the bispecific molecules are shown in Table 27.

All genes are transiently expressed under control of a chimeric MPSVpromoter consisting of the MPSV core promoter combined with the CMVpromoter enhancer fragment. The expression vector also contains the oriPregion for episomal replication in EBNA (Epstein Barr Virus NuclearAntigen) containing host cells.

TABLE 27Amino acid sequences of the bispecific antigen binding molecules Seq IDConstruct Sequence No CD40 (hVH3/hVK2) × FAP (4B9) (4 + 1)with C-terminal VH/VL hVH3_CD40QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 131 VHCH1-VHCH1-GLEWIGRVIPNAGGTSYNQKFKGRVTSTVDTSISTAYMELSRL Fcknob_PGLALA-RSDDTVVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS 4B9 VHKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQGLEWIGRVIPNAGGTSYNQKFKGRVTSTVDTSISTAYMELSRLRSDDTVVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS hVH3_CD40QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 132 VHCH1-GLEWIGRVIPNAGGTSYNQKFKGRVTSTVDTSISTAYMELSRL VHCH1-Fchole_PGLALA-RSDDTVVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS 4B9 VLKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQGLEWIGRVIPNAGGTSYNQKFKGRVTSTVDTSISTAYMELSRLRSDDTVVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPED FAVYYCQQGIMLPPTFGQGTKVEIKhVK2_CD40 DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTFLHWYQQ 133 light chainRPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC CD40 (hVH3/hVK2) ×FAP (4B9) (2 + 1) with C-terminal VH/VL hVH3_CD40QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 134 VHCH1-Fcknob_PGLALA-GLEWIGRVIPNAGGTSYNQKFKGRVTSTVDTSISTAYMELSRL 4B9 VHRSDDTVVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AKGWFGGFNYWGQGTLVTVSShVH3_CD40 QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 135VHCH1-Fchole_PGLALA- GLEWIGRVIPNAGGTSYNQKFKGRVTSTVDTSISTAYMELSRL 4B9 VLRSDDTVVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLP PTFGQGTKVEIK hVK2_CD40DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTFLHWYQQ 133 light chainRPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC CD40 (hVH3/hVK2) ×FAP (4B9) (2 + 2) hVH3_CD40- QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ136 Fc_PGLALA_4B9_VLCH1 GLEWIGRVIPNAGGTSYNQKFKGRVTSTVDTSISTAYMELSRL(charged) RSDDTVVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC hVK2_CD40 LCDVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTFLHWYQQ 137 (charged)RPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 4B9 VHCLEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK 138GLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC CD40 (hVH3/hVK2) ×FAP (4B9) (2 + 1) with C-terminal crossfab hVH3_CD40-Fcknob_PGLALAQVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 139 C-GLEWIGrvipnaggtsynqkfkgRVTSTVDTSISTAYMELSRL term_x4B9_FAP_VL_CH1RSDDTVVYYCARegiywWGQGTTVTVSSASTKGPSVFPLAPSS (charged)KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC hVH3_CD40-Fchole_PGLALAQVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 140 (charged)GLEWIGrvipnaggtsynqkfkgRVTSTVDTSISTAYMELSRLRSDDTVVYYCARegiywWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG +hVK2_CD40DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTFLHWYQQ 137 LC (charged)RPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC +4B9 VHCLEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK 138GLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC CD40 (hVH3/hVK2) ×FAP (4B9) (1 + 1) 4B9-Fcknob_PGLALAEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQ 141APRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGhVH3_CD40-Fchole_PGLALA QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 140(charged) GLEWIGrvipnaggtsynqkfkgRVTSTVDTSISTAYMELSRLRSDDTVVYYCARegiywWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG +hVK2_CD40DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTFLHWYQQ 137 LC (charged)RPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC +4B9 VHCLEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK 138GLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC

Further bispecific antibodies were prepared with the new humanized CD40antibody variants as described in Example 4.2. Specific sequences ofsuch bispecific antibodies are shown in Table 28 below. In particular,different bispecific CD40-FAP antibodies were prepared in 4+1 formatconsisting of four CD40 binding moieties combined with one FAP bindingmoiety as crossover fab fragment fused to the C-terminus of the Fc knobchain (FIG. 15F and FIG. 15G) or in 2+1 format consisting of two CD40binding moieties combined with either one FAP binding moiety ascrossover fab fragment, wherein the VL-CH1 chain is fused at theC-terminus of the Fc knob chain (FIG. 15H) or one FAP binding moiety ascrossover fab fragment, wherein the VH-CL chain is fused at theC-terminus of the Fc knob chain (FIG. 15I).

TABLE 28 Amino acid sequences of bispecific antigen binding moleculesSeq ID Construct Sequence No P1AE0889 CD40 (VH2a/VL2a) ×FAP (28H1) (4 + 1) C-terminal crossfab fusion 28H1 light chainEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 162 cross VHCLGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC VL2a (CD40)DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 243 light chainKPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE (charged)DFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC VH2a (CD40)EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 244 (VHCH1GLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSL charged_VH2aRAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS (CD40) (VHCH1KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ charged)-Fcknob_PGLALA_28H1SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK (VLCH1)SCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGKGLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS C VH2a (CD40)EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 245 (VHCH1GLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSL charged_VH2aRAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS (CD40) (VHCH1KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ charged)-Fchole_PGLALASSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGKGLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGP1AE0890 CD40 (VH2d/VL2a) × FAP (28H1) (4 + 1)C-terminal crossfab fusion 28H1 light chainEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 162 cross VHCLGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC VL2a (CD40)DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 243 light chainKPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE (charged)DFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC VH2d (CD40)EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 246 (VHCH1GLEWVGRVIPNAGGTSYGDSVKGRFTISVDNSKNTAYLQMNSL charged_VH2dRAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS (CD40) (VHCH1KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ charged)-Fcknob_PGLALA_28H1SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK (VLCH1)SCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGKGLEWVGRVIPNAGGTSYGDSVKGRFTISVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS C VH2d (CD40)EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 247 (VHCH1GLEWVGRVIPNAGGTSYGDSVKGRFTISVDNSKNTAYLQMNSL charged_VH2dRAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS (CD40) (VHCH1KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ charged)-Fchole_PGLALASSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGKGLEWVGRVIPNAGGTSYGDSVKGRFTISVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGP1AE2024 CD40 (VH1a/VL1a) × FAP (28H1) (4 + 1)C-terminal crossfab fusion 28H1 light chainEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 162 cross VHCLGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC VL1a (CD40)DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 248 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE (charged)DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC VH1a (CD40)QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 249 (VHCH1)_VH1aSLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL (CD40) (VHCH1)RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS Fcknob_PGLALA_28H1KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ (VLCH1)SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK (charged)SCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQSLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS C VH1a (CD40)QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 250 (VHCH1)_VH1aSLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL (CD40)RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS (VHCH1)-Fchole_PGLALAKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ (charged)SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQSLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGP1AE2302 CD40 (VH1a/VL1a) × FAP (28H1) (2 + 1)C-terminal crossfab fusion 28H1 light chainEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 162 cross VHCLGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC VL1a (CD40)DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 248 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE (charged)DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC VH1a (CD40)QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 251(VHCH1) Fcknob_PGLALA_28H1 SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL(VLCH1) RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS (charged)KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC VH1a (CD40)QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 252 (VHCH1) Fchole_PGLALASLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL (charged)RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE2402CD40 (VH1a/VL1a) × FAP (4B9) (2 + 1) C-terminal crossfab fusion4B9 light chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK 138cross VHCL GLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC VL1a (CD40)DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 248 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE (charged)DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC VH1a (CD40)QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 253(VHCH1) Fcknob_PGLALA_4B9 SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL(VLCH1) RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS (charged)KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC VH1a (CD40)QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 252 (VHCH1) Fchole_PGLALASLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL (charged)RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE2408CD40 (VH1a/VL1a) × FAP (4B9) (2 + 1) C-terminal crossfab fusion4B9 light chain EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQ 254cross VLCH1 APRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC VL1a (CD40)DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 248 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE (charged)DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC VH1a (CD40)QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 255(VHCH1) Fcknob_PGLALA_4B9 SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL(VHCL) RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS (charged)KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC VH1a (CD40)QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 252 (VHCH1) Fchole_PGLALASLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL (charged)RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE2487CD40 (VH1a/VL1a) × FAP (4B9) (2 + 1) C-terminal crossfab fusion4B9 light chain EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQ 254cross VLCH APRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC VL1a (CD40)DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 256 light chainKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC VH1a (CD40)QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 257(VHCH1) Fcknob_PGLALA_4B9 SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL(VHCL) RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC VH1a (CD40)QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 258 (VHCH1) Fchole_PGLALASLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG

5.2 Production of Bispecific Antigen Binding Molecules Targeting CD40and Fibroblast Activation Protein (FAP)

The bispecific antigen binding molecules targeting CD40 and fibroblastactivation protein (FAP) were expressed by transient transfection of HEKcells grown in suspension with expression vectors encoding the 4different peptide chains. Transfection into HEK293-F cells (Invitrogen)was performed according to the cell supplier's instructions usingMaxiprep (Qiagen) preparations of the antibody vectors, F17 medium(Invitrogen, USA), Peipro (Polyscience Europe GmbH) and an initial celldensity of 1-2 million viable cells/ml in serum free FreeStyle 293expression medium (Invitrogen). Cell culture supernatants were harvestedafter 7 days of cultivation in shake flasks or stirred fermenters bycentrifugation at 14000 g for 30 minutes and filtered through a 0.22 μmfilter.

The bispecific antibodies were purified from cell culture supernatantsby affinity chromatography using MabSelectSure-Sepharose™ (GEHealthcare, Sweden) chromatography. Briefly, sterile filtered cellculture supernatants were captured on a MabSelect SuRe resinequilibrated with PBS buffer (10 mM Na₂HPO₄, 1 mM KH₂PO₄, 137 mM NaCland 2.7 mM KCl, pH 7.4), washed with equilibration buffer and elutedwith 25 mM cirate, pH 3.0. After neutralization with 1 M Tris pH 9.0,aggregated protein was separated from monomeric antibody species by sizeexclusion chromatography (Superdex 200, GE Healthcare) in 20 mMhistidine, 140 mM NaCl, pH 6.0. Monomeric protein fractions were pooled,concentrated if required using e.g. a MILLIPORE Amicon Ultra (30KD MWCO)centrifugal concentrator and stored at −80° C. Sample aliquots were usedfor subsequent analytical characterization e.g. by CE-SDS, sizeexclusion chromatography, mass spectrometry and endotoxin determination.

TABLE 29 Production yield and quality of bispecific CD40 antigen bindingmolecules Yield [mg/L] after Protein Purity (by Affinity to A and SEC %CE- Purity (by human Construct purification SDS) % SEC) FAP [nM]P1AE2024 CD40 6.6 mg/L 98.2 97.8 1.2 (VH1a/VL1a) × FAP (28H1) (4 + 1)C-terminal crossfab (knob_VL_CH1) P1AE2302 CD40 12 mg/L 99 99.7 0.3(VH1a/VL1a) × FAP (28H1) (2 + 1) C-terminal crossfab (knob_VL_CH1)P1AE2402 CD40 21 mg/L 96.4 99.2 17.3 (VH1a/VL1a) × FAP (4B9) (2 + 1)C-terminal crossfab (knob_VL_CH1) P1AE2408 CD40 18 mg/L 91.3 95.2 15.3(VH1a/VL1a) × FAP (4B9) (2 + 1) C-terminal crossfab (knob_VH_Ck)P1AE0408 CD40 × FAP 42 mg/L 97.8 96.2 1.5 (28H1) (2 + 1) head-to-tailP1AE0637 CD40 × FAP 9.7 mg/L 98.7 100 0.1 (28H1) (4 + 1) (knob_VL_CH1)P1AE0889 CD40 17 mg/L 96.4 99 nd (VH2a/VL2a) × FAP (28H1) 4 + 1 withC-terminal crossfab P1AE2487 CD40 nd nd 99.2 nd (VH1a/VL1a) × FAP (4B9)(2 + 1) C-terminal crossfab (knob_VH_Ck)

5.3 Characterization of the Bispecific Antibodies Comprising HumanizedCD40 Antibody Variants and FAP 5.3.1 Binding to Human or MouseFAP-Expressing Murine Fibroblast Cells

The binding to cell surface FAP was tested using human fibroblastactivating protein (huFAP) expressing cells NIH/3T3-huFAP clone 19 ormouse fibroblast activating protein (mFAP) expressing cells NIH/3T3-mFAPclone 26 was tested as described in Example 1.4.1. EC₅₀ values asmeasured for some of the bispecific antigen binding molecules comprisinghumanized CD40 antibody variants are shown in Table 7.

5.3.2 Binding to FAP (Surface Plasmon Resonance)

The capacity of the bispecific constructs to bind human FAP was assessedby surface plasmon resonance (SPR). All SPR experiments were performedon a Biacore T200 (Biacore) at 25 ° C. with HBS-EP as running buffer(0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20,(Biacore).

His-tagged human dimeric FAP (recombinant FAP_ECD) was captured on a CMSchip (GE Healthcare) immobilized with anti-His antibody (Qiagen Cat. No.34660) by injection of 500 nM huFAP for 60 s at a flow rate of 10uL/min, 10 nM murine FAP for 20 s at a flow rate of 20 uL/min and 10 nMcynoFAP for 20s at a flow rate of 20 uL/min. Immobilization levels forthe anti-His antibody of up to 18000 resonance units (RU) were used.Following the capture step, the bispecific antibodies as well as controlmolecules were immediately passed over the chip surface at aconcentration ranging from 0.78-100 nM with a flow rate of 30 μL/minutefor 280 s and a dissociation phase of 180 s. Bulk refractive indexdifferences were corrected for by subtracting the response obtained in areference flow cell, where no FAP was immobilized. Avidity wasdetermined using the Langmuir 1:1 curve fitting. For bivalent bindingthe same 1:1 fitting was used leading to an apparent KD value.

TABLE 30 Binding of exemplary bispecific CD40 × FAP antigen bindingmolecules to recombinant human FAP_ECD (Biacore) KD * Ligand (Avidity)ka (1/Ms) kd (1/s) Control 4B9 IgG1 0.08 nM 2.19E+06 1.72E−04 P1AD91394 + 1 with C-terminal 2.7 nM 5.76E+05 1.55E−03 VH/VL fusion P1AE0192 1 +1 crossMab 2.2 nM 6.63E+05 1.45E−03 P1AE0408 2 + 1 head-to-tail 6.0 nM2.91E+05 1.74E−03 format P1AE0637 4 + 1 with C-terminal 7.3 nM 2.82E+052.05E−03 crossFab Note: All K_(D)s are dependent from the specificexperimental conditions.

5.3.3 Binding to CD40 (Surface Plasmon Resonance)

The capacity of the bispecific constructs to bind human CD40 wasassessed by surface plasmon resonance (SPR). All SPR experiments wereperformed on a Biacore T200 (Biacore) at 25° C. with HBS-EP as runningbuffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% SurfactantP20, (Biacore).

In accordance with Example 4.2.7, association was measured by injectionof human CD40 extra cellular domain in various concentrations insolution for 300 sec at a flow of 30 μl/min starting with 300 nM in 1:3dilutions. The dissociation phase was monitored for up to 1200 sec andtriggered by switching from the sample solution to running buffer. Thesurface was regenerated by 60 sec washing with a Glycine pH 2.1 solutionat a flow rate of 30 μl/min. Bulk refractive index differences werecorrected by subtracting the response obtained from a goat anti humanF(ab′)₂ surface. Blank injections are also subtracted (=doublereferencing). For calculation of apparent K_(D) and other kineticparameters the Langmuir 1:1 model was used. The apparent Kd wascalculated using the Biacore™ B4000 evaluation software (version 1.1).

TABLE 31 Binding of exemplary bispecific CD40 × FAP antigen bindingmolecules to recombinant human CD40_ECD (Biacore) Ligand formatdescription KD ka (1/Ms) kd (1/s) P1AE0192 1 + 1 crossMab 3.7 nM2.09E+06 7.77E−03 P1AE0408 2 + 1 head-to-tail format 3.6 nM 2.34E+068.43E−03 P1AE0637 4 + 1 C-terminal crossFab 4.0 nM 1.79E+06 7.22E−03fusion Note: All K_(D)s are dependent from the specific experimentalconditions.

5.3.4 Binding to Human CD40-Expressing Daudi Cells

The binding to cell surface CD40 was tested using Daudi cells, a human Blymphoblast cell line with high expression levels of human CD40 (ATCCCCL-213) as described in Example 1.4.2. Exemplary EC₅₀ values asmeasured for some of the bispecific antigen binding molecules comprisinghumanized CD40 antibody variants are shown in Table 8.

5.3.5 Functional Properties of Bispecific Antigen Binding MoleculeComprising Humanized CD40 Antibody Variants

The functional properties of the bispecific antigen binding moleculescomprising humanized CD40 antibody variants were analyzed in accordanceto the experiments described in Example 2. Exemplary data are providedin Tables 9, 10 or 11 as shown herein before.

1. A bispecific antigen binding molecule, comprising (a) at least oneantigen binding domain capable of specific binding to CD40, and (b) atleast one antigen binding domain capable of specific binding to a targetcell antigen.
 2. The bispecific antigen binding molecule of claim 1,additionally comprising (c) a Fc region composed of a first and a secondsubunit capable of stable association.
 3. The bispecific antigen bindingmolecule of claim 1 or claim 2, wherein the antigen binding domaincapable of specific binding to CD40 binds to a polypeptide comprising,or consisting of, the amino acid sequence of SEQ ID NO:1.
 4. Thebispecific antigen binding molecule of any one of claims 1 to 3, whereinthe antigen binding domain capable of specific binding to a target cellantigen is an antigen binding domain capable of specific binding toFibroblast Activation Protein (FAP).
 5. The bispecific antigen bindingmolecule of any one of claims 1 to 4, wherein the antigen binding domaincapable of specific binding to FAP comprises (a) a heavy chain variableregion (V_(H)FAP) comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:3, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:4, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:5, and a light chain variable region (V_(L)FAP) comprising(iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:6, (v)CDR-L2 comprising the amino acid sequence of SEQ ID NO:7, and (vi)CDR-L3 comprising the amino acid sequence of SEQ ID NO:8, or (b) a heavychain variable region (V_(H)FAP) comprising (i) CDR-H1 comprising theamino acid sequence of SEQ ID NO:11, (ii) CDR-H2 comprising the aminoacid sequence of SEQ ID NO:12, and (iii) CDR-H3 comprising the aminoacid sequence of SEQ ID NO:13, and a a light chain variable region(V_(L)FAP) comprising (iv) CDR-L1 comprising the amino acid sequence ofSEQ ID NO:14, (v) CDR-L2 comprising the amino acid sequence of SEQ IDNO:15, and (vi) CDR-L3 comprising the amino acid sequence of SEQ IDNO:16.
 6. The bispecific antigen binding molecule of any one of claims 1to 5, wherein the antigen binding domain capable of specific binding toFAP comprises (a) a heavy chain variable region (V_(H)FAP) comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO:9, and a lightchain variable region (V_(L)FAP) comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO:10, or (b) a heavy chain variable region(V_(H)FAP) comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO:17, and a light chain variable region (V_(L)FAP) comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO:18.
 7. Thebispecific antigen binding molecule of any one of claims 1 to 6, whereinthe antigen binding domain capable of specific binding to CD40 comprisesa heavy chain variable region (V_(H)CD40) comprising (i) CDR-H1comprising the amino acid sequence of SEQ ID NO:19, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:20, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:21, and a light chainvariable region (V_(L)CD40) comprising (iv) CDR-L1 comprising the aminoacid sequence of SEQ ID NO:22, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO:23, and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO:24.
 8. The bispecific antigen binding molecule ofany one of claims 1 to 6, wherein the antigen binding domain capable ofspecific binding to CD40 comprises a heavy chain variable region(V_(H)CD40) comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:27, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:28, and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:29, and a light chain variable region (V_(L)CD40) comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO:30, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:31, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:32.
 9. The bispecificantigen binding molecule of any one of claims 1 to 8, wherein theantigen binding domain capable of specific binding to CD40 comprises (a)a VH comprising the amino acid sequence of SEQ ID NO:25 and a VLcomprising the amino acid sequence of SEQ ID NO:26, or (b) a VHcomprising the amino acid sequence of SEQ ID NO:33 and a VL comprisingthe amino acid sequence of SEQ ID NO:34.
 10. The bispecific antigenbinding molecule of any one of claims 1 to 7, wherein the antigenbinding domain capable of specific binding to CD40 comprises (i) a heavychain variable region (V_(H)CD40) comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:171, SEQ ID NO:172, SEQID NO:173 and SEQ ID NO:174, and (ii) a light chain variable region(V_(L)CD40) comprising the amino acid sequence selected from the groupconsisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, and SEQ IDNO:178.
 11. The bispecific antigen binding molecule of any one of claims1 to 7, wherein the antigen binding domain capable of specific bindingto CD40 comprises (i) a heavy chain variable region (V_(H)CD40)comprising an amino acid sequence selected from the group consisting ofSEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ IDNO:183 and SEQ ID NO:184, and (ii) a light chain variable region(V_(L)CD40) comprising the amino acid sequence selected from the groupconsisting of SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, and SEQ IDNO:188.
 12. The bispecific antigen binding molecule of any one of claim1 to 7 or 10, wherein the antigen binding domain capable of specificbinding to CD40 comprises (a) a VH comprising the amino acid sequence ofSEQ ID NO:171 and a VL comprising the amino acid sequence of SEQ IDNO:175, or (b) a VH comprising the amino acid sequence of SEQ ID NO:173and a VL comprising the amino acid sequence of SEQ ID NO:177, or (c) aVH comprising the amino acid sequence of SEQ ID NO:174 and a VLcomprising the amino acid sequence of SEQ ID NO:178, or (d) a VHcomprising the amino acid sequence of SEQ ID NO:171 and a VL comprisingthe amino acid sequence of SEQ ID NO:177, or (e) a VH comprising theamino acid sequence of SEQ ID NO:171 and a VL comprising the amino acidsequence of SEQ ID NO:178, or (f) a VH comprising the amino acidsequence of SEQ ID NO:173 and a VL comprising the amino acid sequence ofSEQ ID NO:175, or (g) a VH comprising the amino acid sequence of SEQ IDNO:173 and a VL comprising the amino acid sequence of SEQ ID NO:178, or(h) a VH comprising the amino acid sequence of SEQ ID NO:174 and a VLcomprising the amino acid sequence of SEQ ID NO:175, or (i) a VHcomprising the amino acid sequence of SEQ ID NO:174 and a VL comprisingthe amino acid sequence of SEQ ID NO:177, or (j) a VH comprising theamino acid sequence of SEQ ID NO:171 and a VL comprising the amino acidsequence of SEQ ID NO:176, or (k) a VH comprising the amino acidsequence of SEQ ID NO:172 and a VL comprising the amino acid sequence ofSEQ ID NO:175, or (l) a VH comprising the amino acid sequence of SEQ IDNO:172 and a VL comprising the amino acid sequence of SEQ ID NO:176, or(m) a VH comprising the amino acid sequence of SEQ ID NO:172 and a VLcomprising the amino acid sequence of SEQ ID NO:177, or (n) a VHcomprising the amino acid sequence of SEQ ID NO:172 and a VL comprisingthe amino acid sequence of SEQ ID NO:178, or (o) a VH comprising theamino acid sequence of SEQ ID NO:173 and a VL comprising the amino acidsequence of SEQ ID NO:176, or (p) a VH comprising the amino acidsequence of SEQ ID NO:174 and a VL comprising the amino acid sequence ofSEQ ID NO:176.
 13. The bispecific antigen binding molecule of any one ofclaim 1 to 7 or 10 or 12, wherein the antigen binding domain capable ofspecific binding to CD40 comprises a VH comprising the amino acidsequence of SEQ ID NO:171 and a VL comprising the amino acid sequence ofSEQ ID NO:175.
 14. The bispecific antigen binding molecule of any one ofclaim 1 to 7 or 11, wherein the antigen binding domain capable ofspecific binding to CD40 comprises (a) a VH comprising the amino acidsequence of SEQ ID NO:179 and a VL comprising the amino acid sequence ofSEQ ID NO:185, or (b) a VH comprising the amino acid sequence of SEQ IDNO:180 and a VL comprising the amino acid sequence of SEQ ID NO:185, or(c) a VH comprising the amino acid sequence of SEQ ID NO:181 and a VLcomprising the amino acid sequence of SEQ ID NO:185, or (d) a VHcomprising the amino acid sequence of SEQ ID NO:182 and a VL comprisingthe amino acid sequence of SEQ ID NO:185, or (e) a VH comprising theamino acid sequence of SEQ ID NO:179 and a VL comprising the amino acidsequence of SEQ ID NO:186, or (f) a VH comprising the amino acidsequence of SEQ ID NO:180 and a VL comprising the amino acid sequence ofSEQ ID NO:186, or (g) a VH comprising the amino acid sequence of SEQ IDNO:181 and a VL comprising the amino acid sequence of SEQ ID NO:186, or(h) a VH comprising the amino acid sequence of SEQ ID NO:182 and a VLcomprising the amino acid sequence of SEQ ID NO:186, or (i) a VHcomprising the amino acid sequence of SEQ ID NO:183 and a VL comprisingthe amino acid sequence of SEQ ID NO:187, or (j) a VH comprising theamino acid sequence of SEQ ID NO:183 and a VL comprising the amino acidsequence of SEQ ID NO:188, or (k) a VH comprising the amino acidsequence of SEQ ID NO:184 and a VL comprising the amino acid sequence ofSEQ ID NO:187, or (l) a VH comprising the amino acid sequence of SEQ IDNO:184 and a VL comprising the amino acid sequence of SEQ ID NO:188. 15.The bispecific antigen binding molecule of any one of claim 1 to 7 or 11or 14, wherein the antigen binding domain capable of specific binding toCD40 comprises a VH comprising the amino acid sequence of SEQ ID NO:179and a VL comprising the amino acid sequence of SEQ ID NO:185 or whereinthe antigen binding domain capable of specific binding to CD40 comprisesa VH comprising the amino acid sequence of SEQ ID NO:182 and a VLcomprising the amino acid sequence of SEQ ID NO:185.
 16. The bispecificantigen binding molecule of any one of claims 1 to 7, comprising (i) atleast one antigen binding domain capable of specific binding to CD40,comprising a heavy chain variable region (V_(H)CD40) comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:171, SEQID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:179, SEQ ID NO:180,SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183 and SEQ ID NO:184, and alight chain variable region (V_(L)CD40) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:175, SEQ IDNO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:185, SEQ ID NO:186, SEQID NO:187 and SEQ ID NO:188, and (ii) at least one antigen bindingdomain capable of specific binding to FAP, comprising a heavy chainvariable region (V_(H)FAP) comprising an amino acid sequence of SEQ IDNO:9 and a light chain variable region (V_(L)FAP) comprising an aminoacid sequence of SEQ ID NO:10, or a heavy chain variable region(V_(H)FAP) comprising an amino acid sequence of SEQ ID NO:17 and a lightchain variable region (V_(L)FAP) comprising an amino acid sequence ofSEQ ID NO:18.
 17. The bispecific antigen binding molecule of any one ofclaims 2 to 16, wherein the Fc region is an IgG, particularly an IgG1 Fcregion or an IgG4 Fc region and wherein the Fc region comprises one ormore amino acid substitution that reduces the binding affinity of theantibody to an Fc receptor and/or effector function.
 18. The bispecificantigen binding molecule of any one of claims 2 to 17, wherein the Fcregion is (i) of human IgG1 subclass with the amino acid mutationsL234A, L235A and P329G (numbering according to Kabat EU index), or (ii)of mouse IgG1 subclass with the amino acid mutations D265A and P329G(numbering according to Kabat EU index).
 19. The bispecific antigenbinding molecule of any one of claims 1 to 18, wherein the bispecificantigen binding molecule comprises (a) at least two Fab fragmentscapable of specific binding to CD40 connected to a Fc region, and (b)one antigen binding domain capable of specific binding to FAP connectedto the C-terminus of the Fc region.
 20. The bispecific antigen bindingmolecule of any one of claims 1 to 19, wherein the bispecific antigenbinding molecule comprises (a) at least two Fab fragments capable ofspecific binding to CD40 connected to a Fc region, and (b) a cross-fabfragment capable of specific binding to FAP connected to the C-terminusof the Fc region.
 21. The bispecific antigen binding molecule of any oneof claims 1 to 19, wherein the bispecific antigen binding moleculecomprises four Fab fragments capable of specific binding to CD40. 22.Polynucleotide encoding the bispecific antigen binding molecule of anyone of claims 1 to
 21. 23. An expression vector comprising thepolynucleotide of claim
 22. 24. A host cell comprising polynucleotide ofclaim 22 or the expression vector of claim
 23. 25. A method of producinga bispecific antigen binding molecule, comprising culturing the hostcell of claim 24 under conditions suitable for the expression of thebispecific antigen binding molecule, and isolating the bispecificantigen binding molecule.
 26. A pharmaceutical composition comprisingthe bispecific antigen binding molecule of any one of claims 1 to 21 andat least one pharmaceutically acceptable excipient.
 27. The bispecificantigen binding molecule of any one of claims 1 to 21, or thepharmaceutical composition of claim 26, for use as a medicament.
 28. Thebispecific antigen binding molecule of any one of claims 1 to 21, or thepharmaceutical composition of claim 26, for use (i) in inducing immunestimulation by CD40 expressing antigen-presenting cells (APCs), (ii) instimulating tumor-specific T cell response, (iii) in causing apoptosisof tumor cells, (iv) in the treatment of cancer, (v) in delayingprogression of cancer, (vi) in prolonging the survival of a patientsuffering from cancer, (vii) in the treatment of infections.
 29. Thebispecific antigen binding molecule of any one of claims 1 to 21, or thepharmaceutical composition of claim 26, for use in the treatment ofcancer.
 30. Use of the bispecific antigen binding molecule of any one ofclaims 1 to 21, or the pharmaceutical composition of claim 26, in themanufacture of a medicament for the treatment of cancer.
 31. A method oftreating an individual having cancer comprising administering to theindividual an effective amount of the bispecific antigen bindingmolecule of any one of claims 1 to 21, or the pharmaceutical compositionof claim
 26. 32. The bispecific antigen binding molecule according toany one of claims 1 to 21 or the pharmaceutical composition according toclaim 26 for use in the treatment of cancer, wherein the bispecificantigen binding molecule is administered in combination with achemotherapeutic agent, radiation and/or other agents for use in cancerimmunotherapy.